Prokaryotic Expression Plasmid Research Articles

Prokaryotic Expression and Bioinformatic Analysis of Rv3432c From Mycobacterium tuberculosis

To express the protein enconded by the Rv3432c gene of Mycobacterium tuberculosis (M.tb) in vitro by prokaryotic expression, to analyze the structure of the Rv3432c protein by using bioinformatics software, and to explore for new drug targets against M.tb. The Rv3432c gene was amplified by PCR using the genomic DNA of the inactivated M.tb strain H37Rv as the template and a recombinant plasmid was constructed with the expression vector pET-28a. The expression products were analyzed by SDS-PAGE and purified using affinity chromatography. The biological properties of Rv3432c were analyzed with Protparam, the Pfam online tool, SOMPA, Protscale, TMHMM Signalp 6.0, NetPhos3.1, SUMOsp 2.0, and SWISS-MODEL. pET-28a-Rv3432c recombinant plasmid sequencing results were fully consistent with those of the target gene. SDS-PAGE analysis showed that the fusion protein existed in the form of a soluble protein with a relative molecular mass of about 55×103, which matched the expected size. ProtParam analysis showed that the Rv3432c protein was hydrophilic (showing a GRAVY value of －0.079). Rv3432c was a protein with no transmembrane structural domains or signal peptide. The secondary structure of Rv3432c mainly consisted of random coils (39.78%) and α-helix (39.57%) and was relatively loosely structured. We successfully constructed a prokaryotic expression plasmid of the Rv3432c protein and analyzed its structure using bioinformatics, laying the foundation for further research on the role of Rv3432c in the pathogenesis and progression of tuberculosis as well as the identification of new drug targets against M.tb.

Immunobiological properties and structure analysis of group 13 allergen from Blomia tropicalis and its IgE-mediated cross-reactivity

Blomia tropicalis is an important species of allergenic mite. Structurally related cross-reactive allergens are involved in pathogenesis of clinical symptoms. The present study focused on recombinant allergen rBlo t 13 from B. tropicalis, including investigation of its structure, immunological properties, IgE-mediated cross-reactivity. In this work, the prokaryotic expression plasmids pET-28(a)-Blo t 13, pET-28(a)-Der f 13, and pET-28(a)-Tyr p 13 were constructed, transformed into E. coli Rosetta (DE3) pLysS, and purified by nickel affinity chromatography, respectively. By using ELISA, the IgE-binding rates were detected for rBlo t 13 and its epitope peptides, as well as the cross-reactivity among rBlo t 13, rDer f 13, and rTyr p 13. The tertiary structure of rBlo t 13 was resolved using X-ray diffraction at 2.0 Å resolution. Using IgE-ELISA, the IgE binding rate of rBlo t 13 was 60 % with Blomia tropicalis-positive sera. In the experiments of ELISA for cross-reactivity with rBlo t 13 on solid phase, the inhibition rates were 65 %, 57 % and 63 % for rBlo t 13, rDer f 13, and rTyr p 13, respectively. The structure of Blo t 13 protein contains a β-barrel structure which is composed of 10 β strands and has 2 α helices at the end of the barrel. Comparison of the tertiary structures of rBlo t 13, rDer f 13, and rTyr p 13 revealed that the β-barrel structure is highly conserved, consistent with the alignment of amino acid sequences. We obtained the recombinant protein rBlo t 13, demonstrated its cross-reactivity with Der f 13 and Tyr p 13 due to their structural similarity.

Prokaryotic expression, polyclonal antibody preparation, spatio-temporal expression profile and functional analysis of c-Myc of Helicoverpa armigera (Lepidoptera: Noctuidae)

c-Myc protein encoded by c-Myc (cellular-myelocytomatosis viral oncogene) gene regulates the related gene expression through the Wnt/β-catenin signaling pathway, and has received extensive attention in recent years. The purpose of this study was to express Helicoverpa armigera c-Myc gene (Ha-c-Myc) by using prokaryotic expression system, prepare the polyclonal antibody, examine the spatio-temporal expression profile of Ha-c-Myc, and investigate the possible function of Ha-c-Myc in regulating H. armigera sterol carrier protein-2 (SCP-2) gene expression. The Ha-c-Myc gene was amplified by PCR and cloned into a prokaryotic expression plasmid pET-32a(+). The recombinant plasmid pET-32a-Ha-c-Myc was transformed into Escherichia coli BL21. IPTG was used to induce the expression of the recombinant protein. Protein was purified by Ni2+-NTA column and used to immunize New Zealand rabbits for preparing the polyclonal antibody. The Ha-c-Myc expression levels in different developmental stages (egg, larva, prepupa, pupa, and adult) of H. armigera and different tissues (midgut, fat body, head, and epidermis) of the prepupa were determined by real-time quantitative reverse transcription PCR (qRT-PCR). Ha-c-Myc siRNA was synthesized and transfected into H. armigera Ha cells. The relative mRNA levels of Ha-c-Myc and HaSCP-2 in Ha cells were detected by qRT-PCR. Results showed that the pET-32a-Ha-c-Myc recombinant plasmid was constructed. The soluble Ha-c-Myc protein of about 65 kDa was expressed in E. coli. The polyclonal antibody was prepared. Western blotting analysis suggested that the antibody had high specificity. Enzyme linked immunosorbent assay (ELISA) showed that the titer of the antibody was high. Ha-c-Myc gene expressed at all developmental stages, with high levels in the early and late instars of larva, and the prepupal stage. Tissue expression profiles revealed that Ha-c-Myc expressed in various tissues of prepupa, with high expression level in the midgut, but low levels in the epidermis and fat body. RNAi results showed that the knockdown of Ha-c-Myc expression significantly affected transcription of HaSCP-2, leading to a 50% reduction in HaSCP-2 mRNA expression level. In conclusion, the Ha-c-Myc was expressed through a prokaryotic expression system, and the polyclonal anti-Ha-c-Myc antibody was obtained. Ha-c-Myc may promote the expression of HaSCP-2 and play an important role in the lipid metabolism of H. armigera. These results may facilitate further study on the potential role and function mechanism of Ha-c-Myc in H. armigera and provide experimental data for exploring new targets of green pesticides.

Hp0521 inhibited the virulence of H. pylori 26,695 strain via regulating CagA expression

Hp0521 is the number of cag pathogenicity island (cagPAI) family in Helicobacter pylori (H. pylori, Hp), which encoded Cag2 protein. The aim of this study was to investigate the role of hp0521 on the H. pylori 26,695 strain. We constructed the recombinant prokaryotic expression plasmid pET-32a-hp0521 and pET-32a-hpc0521. Then, we co-cultured the H. pylori wild strain 26,695 and Δhp0521 strain with GES-1 cells to detect CagA protein transport and IL-8 secretion. We found that Δhp0521 mutation increased the expression of cagA, rpoB and promoted the transportation of CagA protein in GES-1 cells. In addition, we also observed that Δhp0521 mutation had no effect on other cagPAI protein stability and the expression of IL-8. Our findings suggested that hp0521 may down-regulated the expression of cagA, rpoB and inhibited the transportation of CagA protein in GES-1 cells and had no effect on growth.

Bioinformatics analysis and prokaryotic expression of Strongyloides stercoralis serine protease inhibitor 1

To predict the structure and antigenic epitope of the Strongyloides stercoralis serine protease inhibitor 1 (Ss-SRPN-1) protein using bioinformatics tools, and to construct prokaryotic expression plasmids for expression of recombinant Ss-SRPN-1 protein, so as to provide the basis for unraveling the function of the Ss-SRPN-1 protein. The amino acid sequence of the Ss-SRPN-1 protein was downloaded from the NCBI database, and the physicochemical properties, structure and antigenic epitopes of the Ss-SRPN-1 protein were predicted using bioinformatics tools, including ExPASy, SWISS-MODEL and Protean. Primers were designed according to the nucleotide sequences of Ss-SRPN-1, and the Ss-SRPN-1 gene was amplified, cloned and sequenced with genomic DNA extracted from the infective third-stage larvae of S. stercoralis as a template. The Ss-SRPN-1 protein sequence was cloned into the pET28a (+) expression vector and transformed into Escherichia coli BL21 (DE) cells for induction of the recombinant Ss-SRPN-1 protein expression. The recombinant Ss-SRPN-1 protein was then purified and identified using Western blotting and mass spectrometry. Bioinformatics analysis showed that the Ss-SRPN-1 protein, which was composed of 372 amino acids and had a molecular formula of C1948H3046N488O575S16, was a stable hydrophilic protein, and the subcellular localization of the protein was predicted to be extracellular. The Ss-SRPN-1 protein was predicted to contain 11 dominant B-cell antigenic epitopes and 20 T-cell antigenic epitopes. The Ss-SRPN-1 gene with a length of 1 119 bp was successfully amplified, and the recombinant plasmid pET28a (+)/Ss-SRPN-1 was constructed and transformed into E. coli BL21(DE) cells. The expressed recombinant Ss-SRPN-1 protein had a molecular weight of approximately 43 kDa, and was characterized as a Ss-SRPN-1 protein. The recombinant Ss-SRPN-1 protein has been expressed successfully, and this recombinant protein may be a potential vaccine candidate against strongyloidiasis.

Comparative analysis of the anticoagulant activities and immunogenicity of HSC70 and HSC70TKD of Haemaphysalis flava

BackgroundHaemaphysalis flava is a hematophagous ectoparasite that acquires the nutrition needed for development and reproduction by sucking blood and digesting the blood meal. During blood-sucking and blood-meal digestion, the prevention of blood coagulation is important for this tick. Previous studies have shown that heat shock cognate 70 (HSC70) protein has certain anticoagulant activities, but its immunogenicity remains unclear. Also, whether the mutation of individual bases of the TKD-like peptide of HSC70 through the overlap extension method can change its anticoagulant activities and immunogenicity remains to be investigated.MethodsThe gene encoding the HSC70 protein was cloned from a complementary DNA library synthesized from H. flava. The coding gene of the TKD-like peptide of HSC70 was mutated into a TKD peptide coding gene (HSC70TKD) using the overlap extension method. Escherichia coli prokaryotic expression plasmids were constructed to obtain the recombinant proteins of HSC70 (rHSC70) and HSC70TKD (rHSC70TKD). The purified rHSC70 and rHSC70TKD were evaluated at different concentrations for anticoagulant activities using four in vitro clotting assays. Emulsifying recombinant proteins with complete and incomplete Freund’s adjuvants were subcutaneously immunized in Sprague Dawley rats. The serum antibody titers and serum concentrations of interferon-gamma (IFN-γ) and interleukin-4 (IL-4) were detected using an indirect enzyme-linked immunosorbent assay to assess the immunogenicity of rHSC70 and rHSC70TKD.ResultsThe open reading frame of HSC70 was successfully amplified and found to have a length of 1958 bp. The gene encoding the TKD-like peptide of HSC70 was artificially mutated, with the 1373-position adenine (A) of the original sequence mutated into guanine (G), the 1385-position cytosine (C) mutated into G and the 1386-position G mutated into C. rHSC70 and rHSC70TKD that fused with His-tag were obtained using the expression plasmids pET-28a-HSC70 and pET-28a-HSC70TKD, respectively. rHSC70 and rHSC70TKD prolonged the thrombin time (TT) and reduced the fibrinogen (FIB) content in the plasma, but did not affect the prothrombin time (PT) or activated partial thromboplastin time (APTT) when compared to the negative control. Interestingly, the ability of rHSC70TKD to prolong the TT and reduce the FIB content in the plasma was better than that of rHSC70. The specific antibody titers of both rHSC70 and rHSC70TKD in rat serum reached 1:124,000 14 days after the third immunization. The serum concentration of IFN-γ in the rHSC70TKD group was higher than that in the rHSC70 group. The rHSC70 group has the highest serum concentration of IL-4, and the serum concentration of IL-4 in the rHSC70TKD group was higher than that in the negative group.ConclusionsrHSC70 and rHSC70TKD exhibited anticoagulant activities by prolonging the TT and reducing the FIB content in vitro. rHSC70TKD had better anticoagulant activities than rHSC70. Both rHSC70 and rHSC70TKD had good immunogenicity and induced humoral and cellular immunity.

Studies on HBcAg-rBlo t 5-21 Fusion Protein Vaccine That Alleviates Blomia tropicalis Airway Inflammation.

In tropical and subtropical areas, allergens from the dust mite species Blomia tropicalis are common causes of allergic rhinitis and asthma. Blomia tropicalis has two main allergens: Blo t 5 and Blo t 21. To generate a chimeric virus-like particle containing HBcAg, Blo t 5 and Blo t 21 that can treat allergies caused byBlomia tropicalis. To produce allergic asthma in mice, prokaryotic expression and purification of Blomia tropicalis allergens rBlo t 5, rBlo t 21, and recombinant fusion allergen rBlo t 5-21 were utilized in the study. We created a hepatitis B core antigen (HBcAg) and rBlo t 5-21 fusion prokaryotic expression plasmid. HBcAg-rBlo t 5-21 was purified after expression and tested by transmission electron microscopy (TEM). Furthermore, the protein HBcAg-rBlo t 5-21 was employed as a protein vaccination. In allergy-induced mouse model experiments, the fusion allergen rBlo t 5-21 was more effective than the individual allergens rBlo t 5 and rBlo t 21 at inducing allergy. We found that vaccinating allergic mice with the recombinant fusion protein vaccine HBcAg-rBlo t 5-21 alleviated allergy symptoms elicited by the rBlo t 5-21 allergen. Vaccination with HBcAg-rBlo t 5-21 resulted in a decrease in total serum IgE levels, suppression of anaphylaxis, and reduction of inflammatory cell infiltration into lung tissue as compared to the PBS group. HBcAg-rBlo t 5-21, a protein vaccine containing both the hepatitis B core antigen and the Blomia tropicalis fusion allergen rBlo t 5-21, could be a suitable vaccination for preventing allergy disorders caused by Blomia tropicalis.

Cloning, Exogenous Expression and Function Analysis of Interferon-γ from Gadus macrocephalus.

Interferon γ (IFN–γ) is now considered to be one of the key molecules in the regulation of innate and adaptive immunity. The function of IFN–γ is best described in humans, but less of IFN–γ in fish species has been described at protein level. In the present study, IFN–γ from Gadus macrocephalus (GmIFN–γ) has been examined in terms of bioinformatics, prokaryotic expression, yeast expression, antiviral activity and immune regulatory function. The cDNA of GmIFN–γ contains an open reading frame of 570 nucleotides, coding 189 amino acids. The mature protein contains a nuclear localization signal motif and an obvious IFN–γ signature sequence at the C-terminal. GmIFN–γ is very similar to that of Atlantic cod, with homology up to 89.89%, but less than 32% to other species. GmIFN–γ can be detected in the gills, spleen, intestine, brain and kidney. Interestingly, during early development, a strong signal of GmIFN–γ was not detected until 40 days post hatching. Prokaryotic expression plasmid pET–32a–GmIFN–γ was constructed, and the expression products in BL21 were confirmed by Mass Spectrometry. Meanwhile, the plasmid pGAPZA–GmIFN–γ with Myc tag was constructed and transmitted into Pichia pastoris yeast GS115, and the products were tested using Western blot. The purified GmIFN–γ from either BL21 or yeast has a strong antivirus (Spring viremia of carp virus) effect. The vector of pcDNA3.1–GmIFN–γ was expressed in EPC cell lines; high transcript levels of MHC class I chain-related protein A (MICA) gene were detected; and the exogenous GmIFN–γ protein could also induce MICA expression, indicating that GmIFN–γ could stimulate immune response. The yeast GS115 with GmIFN–γ protein, which is an inclusion body, was given to zebrafish orally, and the transcript of zebrafish IFN–γ was upregulated significantly; however, genes of the interferon type–I signal pathway were not well stimulated.

Mutations in SARS-CoV-2 nucleocapsid in variants of concern impair the sensitivity of SARS-CoV-2 detection by rapid antigen tests

Rapid antigen tests (RATs) are used as convenient SARS-CoV-2 tests to minimize infection risks in the private and public domain (e.g., access to shops, concerts, sports, and other social events). RATs are: however, less sensitive than quantitative reverse transcription Polymerase chain reaction (RT-qPCR) assays; hence, samples with low viral loads may be misdiagnosed. Reports on the ability of RATs to detect SARS-CoV-2 variants of concern (VOCs) Delta and Omicron are often only qualitative. We, therefore, examined the analytical sensitivities of four different RATs for the detection of both full virus and recombinant proteins of relevant VOCs. Since most RATs are based on the detection of the SARS-CoV-2 nucleocapsid protein (N-protein), we constructed multiple N-protein mutants (mirroring specific amino acid exchanges of VOC N-proteins) using prokaryotic expression plasmids and site-directed PCR mutagenesis. Testing of recombinant proteins by four RATs revealed amino acid substitutions R203K and R203M, are critical for the sensitivity of some RATs. Interestingly, R203M mutation completely abrogated antigen detection even at high protein concentrations in the Delta variant. As a proof-of-concept study, we show that one or two specific amino acid changes in the N-protein can negatively impact the analytical sensitivity of RATs. Hence, antibodies used in such lateral flow assays should be optimized and target preferentially more conserved regions of N-protein.

Prokaryotic expression and purification of nucleoprotein of Guertu virus and its establishment of ELISA detection method

Objective: To obtain purified protein antigen of guertu virus (GTV) nucleoprotein (NP) and establish a rapid and accurate enzyme-linked immunosorbent assay (ELISA) method for detection of GTV antibody. Methods: Codon optimized GTV NP encoding genes were synthesized, cloned into the pet32a (+) vector, and recombinant expression plasmids were constructed and transformed into BL21 (DE3). Recombinant protein (rNP) obtained from the optimized expression were purified over a Ni column and identified by SDS-PAGE and Western blot. The purified protein was used as the antigen to optimize the reaction conditions, and an indirect ELISA assay for GTV IgG antibody was developed and optimized, which was evaluated and initially applied. Results: The prokaryotic expression plasmid pet32a-NP was successfully constructed, the recombinant protein was highly expressed in E. coli in the form of inclusion bodies, the size was about 44 kD, and the results of Western blot indicated that the recombinant protein had good antigenicity with GTV positive serum. The optimized ELISA (GTV-rNP-iELISA) established in this study showed strong specificity, high sensitivity, and the coefficient of variation within and between batches is less than 10%, and has good repeatability; the detection results are consistent with the IFA detection results. Using the established ELISA method to detect 162 sheep sera from some regions of Xinjiang in 2017-2019, the total positive rate of antibodies was 39.8%. Conclusions: The GTV NP antibody detection ELISA method has good sensitivity, reproducibility, and specificity and has the potential to be a powerful tool for the diagnosis and serological investigation of GTV infection.

Development of an Indirect ELISA to Detect African Swine Fever Virus pp62 Protein-Specific Antibodies.

African swine fever (ASF) is a highly detrimental viral disease caused by African swine fever virus (ASFV). The occurrence and prevalence of this disease have become a serious threat to the global swine industry and national economies. At present, the detection volume of African swine fever is huge, more sensitive and accurate detection techniques are needed for the market. pp62 protein, as a protein in the late stage of infection, has strong antigenicity and a high corresponding antibody titer in infected pigs. In this study, the CP530R gene was cloned into expression vector pET-28a to construct a prokaryotic expression plasmid, which was induced by IPTG to express soluble pp62 protein. Western blot analysis showed that it had great reactivity. Using the purified recombinant protein as an antigen, an indirect ELISA method for detecting ASFV antibody was established. The method was specific only to ASFV-positive serum, 1:1600 diluted positive serum could still be detected, and the coefficients of variation (CV) of the intra assay and inter assay were both <10%. It turns out that the assays had excellent specificity, sensitivity, and repeatability. This provides an accurate, rapid, and economical method for the detection of ASFV antibody in clinical pig serum samples.

Preparation of antibodies against TXR1 and construction of a new DNA tumor vaccine

BackgroundTaxol-resistance gene 1 (TXR1) is closely correlated with the paclitaxel resistance in the cancer chemotherapy. However, due to the lack of monoclonal antibodies (mAbs) with strong specificity and high sensitivity, little information is found about TXR1 target-related tumor therapy. MethodsWe developed an TXR1 recombinant DNA vaccine by inserting TXR1 DNA sequence into lysosome-associated membrane protein 1 (LAMP1). Adaptive immune responses were assessed by indirect enzyme-linked immunosorbent assay (ELISA), Enzyme-linked immunospot test (ELISpot), and cytotoxic T-lymphocyte (CTL) cytotoxicity. ResultsThe pGEX4T-1-TXR1 reconstructed prokaryotic expression plasmid was constructed for producing high-purity TXR1 protein. Subsequently, a total of four mAbs for TXR1 and two PcAbs were successfully constructed and identified. We further found that TXR1 was highly expressed in breast cancer tissue than normal controls. Therefore, we constructed four tumor vectors, pVAX1-LAMP/TXR1, pVAX1-LAMP, pVAX1/TXR1 and pVAX1, for immunization. After three times of immunization, ELISpot data showed that single peptide 6,9,11 could stimulate T cells secreting IFN-γ in pVAX1-LAMP/TXR1 group. Moreover, the number of specific T cells and immune response effects significantly increased comparing to the pVAX1-LAMP control group. In addition, cytotoxicity showed that when the effect to target ratio was 40:l the killing effect of pVAX1-LAMP/TXR1 group was significantly higher than the pVAX1-TXR1 group. ConclusionOur results provides new evidence for the TXR1 related tumor immunology and aids the early prevention of cancer.

Decrescent role of recombinant HSP60 antibody against atherosclerosis in high-cholesterol diet immunized rabbits.

Atherosclerosis is the main cause of cardiovascular disease (CVD) which has a key role in the development of coronary artery disease (CAD). Based on clinical studies, HSP60 is the only HSP that can cause atherosclerosis. In this paper, the expression level of HSP60 and the pathogenic degree of its cloned part was investigated in atherosclerosis condition. After the designation of the specific primers for HSP60, PCR was done by the Pfu enzyme. Subsequently, the PCR products were cloned into a prokaryotic expression vector pET-28a. The resultant recombinant vector was transferred in BL21 and purified. Purification of protein was done by the Nickel affinity column. After confirmation of Western blotting and HSP60 protein purification, purified protein concentration was measured by the Bradford method, and purity was analyzed by SDS PAGE 12%. New Zealand rabbits were tested as an animal model. At the next step, the recombinant protein was injected into the animal model that was on a fatty diet. The prokaryotic expression plasmid pET28a-hps60 was successfully constructed, the HSP60 protein was expressed and purified in Escherichia coli BL21 (DE3). We found that the rabbit that was receiving the recombinant vaccine with the fatty diet showed a lower amount of fat deposition at the media endothelial level than the rabbit which received only the fatty diet. Taking recombinant protein concomitant with a fatty diet, causes betterment of atherosclerosis via decreasing aggregation of cholesterol and thickness of the endothelial media.

Generation and immunogenicity evaluation of Senecavirus A virus-like particles

To develop Senecavirus A (SVA) virus-like particles (VLPs), a recombinant prokaryotic expression plasmid pET28a-SVA-VP031 was constructed to co-express SVA structural proteins VP0, VP3 and VP1, according to the genomic sequence of the field isolate CH-FJ-2017 after the recombinant proteins were expressed in E .coli system, and purified by Ni+ ion chromatographic method. The SVA VLPs self-assemble with a high yield in vitro buffer. A typical VLPs with an average diameter of 25-30 nm which is similar to native virions by using TEM detection. Animals immunized by SVA VLPs shown that the VLPs induced high titers neutralizing antibodies in Guinea pigs. This study indicated that the VLPs produced with co-expressing SVA structural proteins VP0, VP3 and VP1 in prokaryotic system is a promising candidate and laid an important foundation for the development of a novel SVA VLPs vaccine.

Construction and Expression of Eukaryotic and Prokaryotic Recombinant Vectors of Pepck and Gp63 Dominant Epitopes of Leishmania infantum

To construct eukaryotic and prokaryotic recombinant vectors containing Pepck- Gp63 and to achieve protein expression by selecting the dominant epitope genes of Pepck and Gp63 of Leishmania infantum. The secondary structure and HLA epitopes of phosphoenolpyruvate carboxylase (PEPCK) were predicted by in silico analysis, and the dominant epitopes were picked out. According to the analysis results of glycoprotein of 63×10 3(GP63) epitopes identified by the same method in our laboratory, the dominant epitope genes of Pepck and Gp63 were used to construct pET32a- Pepck- Gp63 and pVAX1- Pepck- Gp63 by overlapping PCR and enzyme reaction. Then, for protein expression, the prokaryotic vectors were transfected into E.coil while the eukaryotic vectors were transfected into NIH3T3 cells by liposome transfection. There were multiple dominant epitopes in Pepckand there were Pepck-Gp63 sequences in the polyclonal site of expression vector. The expression of Pepck-Gp63 in E.coil appeared in inclusion form and led to 74 kDa band in SDS-PAGE. The immunofluorescence results of NIH3T3 cells transfected by pVAX1- Pepck-Gp63 were positive. The recombinant prokaryotic expression plasmids pET32a- Pepck-Gp63 and eukaryotic expression plasmids pVAX1- P epck -Gp63 were successfully constructed, and it was shown that the recombinant plasmids were able to express the corresponding target proteins in E. coli and NIH3T3 cells, respectively, providing a preliminary experimental basis for the subsequent study of immunization strategies.

Functional expression and purification of recombinant full-length human ATG7 protein with HIV-1 Tat peptide in Escherichia coli

The human autophagy-related protein ATG7 (hATG7), an E1-like ubiquitin enzyme, activates two ubiquitin-like proteins, LC3 (Atg8) and Atg12, and promotes autophagosome formation. While hATG7 plays an essential role for the autophagy conjugation system, the production of full-length functional hATG7 in bacterial systems remains challenging. Previous studies have demonstrated that the HIV-1 virus-encoded Tat peptide (‘GRKKRRQRRR’) can increase the yield and solubility of heterologous proteins. Here, functional full-length hATG7 was expressed using the pET28b-Tat expression vector in the Escherichia coli BL21 (DE3) strain. Recombinant hATG7 protein aggregated as inclusion bodies while expressed with widely used prokaryotic expression plasmids. In contrast, the solubility of Tat-tagged hATG7 increased significantly with prolonged time compared to Tat-free hATG7. The recombinant proteins were purified to >90% homogeneity under native conditions with a single step of affinity chromatography purification. The results of in vitro pull-down and LC3B–I lipidation assays showed that Tat-tagged hATG7 directly interacted with LC3B–I and promoted LC3B–I lipidation, suggesting that Tat-tagged hATG7 has significant catalytic activity. Overall, this study provides a novel method for improving the functional expression of full-length hATG7 in bacterial systems by fusion with the Tat peptide, a process which may be applied in future studies of hATG7 structure and function.

Expression, Purification, and Bioactivity of a Soluble Recombinant Ovine Interferon-tau in Escherichia Coli.

IntroductionOvine interferon-tau (oIFN-τ) is a newly discovered type I interferon. This study used biochemical techniques to transform the oIFN-τ gene into Escherichia coli to obtain the mass and soluble expression of the recombinant protein.Material and MethodsFirst, total RNA was extracted from fresh sheep embryonic tissues with TRIzol reagent and then used as a template to reverse transcribe and amplify the mature oIFN-τ gene with RT-PCR. The amplified product was next digested with the HindIII and XhoI restriction enzymes and inserted into the pET-32a(+) vector to construct the prokaryotic expression plasmid. The corrected in-frame recombinant plasmid, pET-32a(+)-oIFN-τ, was transformed into E. coli Rosetta (DE3) competent cells. After induction with isopropyl-beta-D-thiogalactopyranoside (IPTG), the recombinant protein was detected in bacteria. Finally, the bacteria were lysed by sonication, and the recombinant protein was purified by nickel affinity chromatography and DEAE anion exchange chromatography.ResultsThe protein was confirmed to be oIFN-τ, which mainly existed in the soluble lysate fraction, as proven by SDS-PAGE and Western blot assays.ConclusionPurified IFN-τ exists mostly in a soluble form, and its anti-vesicular stomatitis virus (VSV) activity reached 7.08×10(6)IU/mL.

Isolation, purification, gene cloning and expression of antifungal protein from Bacillus amyloliquefaciens MG-3

The antifungal protein MG-3A was isolated from Bacillus amyloliquefaciens MG-3, and was purified and identified. The results showed that antifungal protein MG-3A was likely a serine protease with a molecular weight of ~48 kDa. The serine protease exhibited a broad antifungal spectrum and effectively extended the shelf-life of loquat fruit up to 25 d. The antifungal protein MG-3A showed good stabilities to temperature, pH and protease K. Primers were designed according to the mass spectrum of antifungal protein and the comparison with proteins in the NCBI database. The serine protease gene MG-3A from B. amyloliquefaciens genome was isolated and cloned using PCR. The prokaryotic expression plasmid pET28a-MG-3A was constructed and used to express the antimicrobial protein in vitro. The SDS-PAGE results showed that the recombinant protein expressed in Escherichia coli BL21 (DE3) was highly soluble. Affinity chromatography was used to purify the recombinant protein and its antifungal activity was evaluated.

Molecular cloning and expression of hosphoenolpyruvate carboxykinase dominant epitope in Leishmania infantum

Objective Based on the (phosphoenolpyruvate carboxykinase, PEPCK) dominant epitopes gene of Leishmania infantum that has been analyzed and selected, we construct the corresponding recombinant prokaryotic expression vector to obtain the recombinant protein, construct the corresponding recombinant eukaryotic expression vector and verify the expression of eukaryotic vector in NIH3T3 cells, which laid the foundation for the subsequent vaccination and infection of animal experiment. Methods Based on the PEPCK dominant epitopes gene sequence, the recombinant prokaryotic and eukaryotic expression plasmid pET32a- PEPCK and pVAX1- EPEPCK were constructed by PCR reaction and enzyme reaction and were respectively transfected into E. coli and NIH3T3 cells of mice for expression. The expression of prokaryotic recombinant plasmid and the prokaryotic expressed protein purified by HisTrap affinity column were detected through SDS-PAGE and Western-Blot, and the expression of pVAX1-E PEPCK was verified by immunofluorescence. Results The correct sequencing results of the recombinant prokaryotic and eukaryotic vectors showed that the construction of recombinant vectors was successful. The positive results of 48.08 kD of recombinant protein were detected in SDS-PAGE and Western Blot, and immunofluorescence results of NIH3T3 cells transfected with eukaryotic recombinant vectors showed positive results. Conclusion We successfully constructed the recombinant prokaryotic and eukaryotic expression vectors: pET32a- PEPCK and pVAX1-E PEPCK , successfully got the recombinant proteins that followed by proteinic purification and confirmed the expression of pVAX1-E PEPCK in NIH3T3 cells, which is the basis for experiments where animals are vaccinated with a DNA prime-protein boost vaccines. 摘要：目的 基于前期分析并选取的婴儿利什曼原虫 PEPCK 的优势表位基因, 构建相应重组原核表达载体以获得 重组蛋白, 构建相应重组真核表达载体并验证真核载体在 NIH3T3 细胞中的表达, 为后续动物的免疫和感染实验备下 基础。 方法 根据 PEPCK 优势表位基因序列, 经 PCR 反应及酶切连接构建重组原核与真核表达质粒 pET32a- PEPCK 和 pVAX1-E PEPCK 并分别转染至 E. coli 和 NIH3T3 细胞中进行表达。采用 SDS-PAGE 电泳和 Western Blot 鉴定原核重 组质粒在大肠杆菌中的表达和原核表达蛋白被镍柱纯化后的情况, 采用免疫荧光实验验证真核重组质粒在细胞中的 表达。 结果 重组原核与真核载体的正确测序结果表明重组载体构建成功。SDS-PAGE 电泳和 Western Blot 的结果 显示, 大肠杆菌中表达的原核重组蛋白以及纯化后的蛋白在 48.08 kD 处存在条带, 转染了真核重组载体的 NIH3T3 细胞 的免疫荧光结果呈阳性。 结论 成功构建了 PEPCK 优势表位基因的重组原核和真核表达载体: pET32a- PEPCK 和 pVAX1-E PEPCK , 成功表达相应的重组蛋白并纯化并验证了 pVAX1-E PEPCK 在 NIH3T3 细胞中的表达, 为后续 DNA 疫苗初次免疫和蛋白疫苗加强免疫的动物实验备下基础。

Effects of diclazuril on the expression of enolase in second-generation merozoites of Eimeria tenella

Eimeria tenella is an obligate intracellular parasite of the chicken cecum; it brings huge economic loss to the chicken industry. Enolase is a multifunctional glycolytic enzyme involved in many processes of parasites, such as infection and migration. In this study, the effect of diclazuril on the expression of enolase in second-generation merozoites of E. tenella (EtENO) was reported. The prokaryotic expression plasmid pET-28a-EtENO was constructed and transformed into Escherichia coli BL21 (DE3). Then, it was subjected to expression under the induction of isopropyl-β-D-1-thiogalactopyranoside. The expressed products were identified and purified. The purified EtENO protein was used for antibody preparation. The EtENO mRNA and protein expression levels were analyzed via real-time PCR and Western blotting. Localization of EtENO on the merozoites was examined by immunofluorescence technique. The mRNA and protein expression levels of EtENO were decreased by 36.3 and 40.36%, respectively, by diclazuril treatment. EtENO distributed in the surface, cytoplasm, and nucleus of the infected/control group. With diclazuril treatment, it was significantly reduced in the surface and cytoplasm and even disappeared in the nucleus of the infected/diclazuril group. These observations suggested that EtENO may play an important role in mechanism of diclazuril anticoccidial action and be a potential drug target for the intervention with E. tenella infection.