Expression Plasmid pET-28a Research Articles

Optimizing the Metal Bioreduction Process in Recombinant Shewanella azerbaijanica Bacteria: A Novel Approach via mtrC Gene Cloning and Nitrate-Reducing Pathway Destruction.

Environmental pollution is growing every day in terms of the increase in population, industrialization, and urbanization. Shewanella azerbaijanica is introduced as a highly potent bacterium in metal bioremediation. The mtrC gene was selected as a cloning target to improve electron flux chains in the EET (extracellular electron transfer) pathway. Using the SDM (site-directed mutagenesis) technique, the unique gene assembly featured the mtrC gene sandwiched between two napD/B genes to disrupt the nitrate reduction pathway, which serves as the primary metal reduction competitor. Shew-mtrC gene construction was transferred to expression plasmid pET28a (+) in the expression host bacteria (E. coli BL21 and S. azerbaijanica), in pUC57, cloning plasmid, which was transferred to the cloning host bacteria E. coli Top10 and S. azerbaijanica. All cloning procedures (i.e., synthesis, insertion, transformation, cloning, and protein expression) were verified and confirmed by precise tests. ATR-FTIR analysis, CD, western blotting, affinity chromatography, SDS-PAGE, and other techniques were used to confirm the expression and structure of the MtrC protein. The genome sequence and primers were designed according to the submitted Shewanella oneidensisMR-1 genome, the most similar bacteria to this native species. The performance of recombinant S. azerbaijanica bacterium in metal bioremediation, as sustainable strategy, has to be verified by more research.

Role of Cherry Valley duck IRF1 mediated signal pathway in host anti-duck Tembusu virus

China is the country with the largest amount of duck breeding as well as duck meat and egg production. In recent years, the emergence and spread of duck Tembusu virus (DTMUV) has become one of the important factors in reducing the amount of duck slaughter, which seriously endangers the duck breeding industry in our country. In-depth research on the mechanism of duck innate immunity facilitates the exploration of new models for the treatment of DTMUV infection. IRF1 can induce the expression of many antiviral immune factors in the animal organism and play an important role in the innate immune response. In this study, we used interfering RNA to knock down the IRF1 gene in DEF cells and then the cells were infected with DTMUV. We found that knockdown of IRF1 promoted DTMUV replication at an early stage and caused downregulation of the expression of several major pattern recognition receptors (PRRs), interleukins (IL), interferons (IFN), antiviral proteins, and MHC molecules by assay, showing that the duIRF1-mediated signaling pathway plays an extremely important role in DTMUV-induced host innate immunity. In addition, we constructed the recombinant expression plasmid pET32a(+)-duIRF1-His, and finally prepared the polyclonal antibody of duIRF1 with good specificity, hoping to provide a detection means for research on the mechanism of IRF1 in innate immunity in our laboratory and in this field.

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.

Prediction of superantigen active sites and clonal expression of staphylococcal enterotoxin-like W

Objective: The docking and superantigen activity sites of staphylococcal enterotoxin-like W (SElW) and T cell receptor (TCR) were predicted, and its SElW was cloned, expressed and purified. Methods: AlphaFold was used to predict the 3D structure of SElW protein monomers, and the protein models were evaluated with the help of the SAVES online server from ERRAT, Ramachandran plot, and Verify_3D. The ZDOCK server simulates the docking conformation of SElW and TCR, and the amino acid sequences of SElW and other serotype enterotoxins were aligned. The primers were designed to amplify selw, and the fragment was recombined into the pMD18-T vector and sequenced. Then recombinant plasmid pMD18-T was digested with BamHⅠand Hind Ⅲ. The target fragment was recombined into the expression plasmid pET-28a(+). After identification of the recombinant plasmid, the protein expression was induced by isopropyl-beta-D- thiogalactopyranoside. The SElW expressed in the supernatant was purified by affinity chromatography and quantified by the BCA method. Results: The predicted three-dimensional structure showed that the SElW protein was composed of two domains, the amino-terminal and the carboxy-terminal. The amino-terminal domain was composed of 3 α-helices and 6 β-sheets, and the carboxy-terminal domain included 2 α-helices and 7 antiparallel β-sheets composition. The overall quality factor score of the SElW protein model was 98.08, with 93.24% of the amino acids having a Verify_3D score ≥0.2 and no amino acids located in disallowed regions. The docking conformation with the highest score (1 521.328) was selected as the analysis object, and the 19 hydrogen bonds between the corresponding amino acid residues of SElW and TCR were analyzed by PyMOL. Combined with sequence alignment and the published data, this study predicted and found five important superantigen active sites, namely Y18, N19, W55, C88, and C98. The highly purified soluble recombinant protein SElW was obtained with cloning, expression, and protein purification. Conclusions: The study found five superantigen active sites in SElW protein that need special attention and successfully constructed and expressed the SElW protein, which laid the foundation for further exploration of the immune recognition mechanism of SElW.

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.

Expression and functional identification of recombinant SARS-CoV-2 receptor binding domain (RBD) from E. coli system

The receptor binding domain (RBD) of SARS-CoV-2 is located in the C-terminal of S1 subunit of the spike (S) protein which is responsible for recognizing and binding to the angiotensin-converting enzyme 2 (ACE2) receptor. The DNA encoding the SARS-CoV-2 RBD was inserted into pET-28a (+) to construct expression plasmid pET-28a (+)/RBD. The desired RBD protein was produced in E. coli Rosetta (DE) and purified by a Ni-NTA column. The recombinant RBD was analyzed by SDS-PAGE and Western blot. The flow cytometry analysis indicated that the recombinant RBD is capable of binding to human ACE2 (hACE2) in the ACE2-overexpressed HEK293A-hACE2 cells. Our results demonstrated that recombinant RBD expressed in E. coli Rosetta (DE) strain has bioactivities and can be used as an antigen for diagnosis and as a tool for the development of novel anti-viral drugs against SASR-CoV-2.

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。

Cloning and sequence analysis of leptin receptor overlapping transcript-like 1 gene from Dermatophagoides farinae

To obtain the leptin receptor overlapping transcript-like 1 encoding gene (LepROTL1 gene) from Dermatophagoides farina, investigate the molecular characteristics of the gene and construct a prokaryotic expression vector to express this gene. The LepROTL1 gene-encoding sequence fragments were captured based on the transcriptome sequencing results, and the full-length gene fragments were amplified from total RNA of D. farinae using a RT-PCR assay, and used to construct the expression plasmid pET28a(+)-LepROTL1, followed by sequencing. The plasmid was transformed into E. coli BL21 (DE3) T1R for the induction of IPTG expression. The expression product was characterized by SDS-PAGE and Western blotting. Bioinformatics analyses were performed to analyze the sequence and the molecular characteristics of its encoded protein. The amplification products of the RT-PCR assay showed a clear band on agarose gel electrophoresis, and sequencing analysis of the pET28a(+)-LepROTL1 plasmid showed 417 bp in length of the coding gene from the start codon ATG to the termination codon TAA. Following the plasmid transformation into E. coli and induction with IPTG, a specific band was seen on SDS-PAGE, indicating successful expression. Bioinformatics analysis showed that the LepROTL1 gene-encoded protein was composed of 134 amino acids, and had a relative molecular weight of 14 378.13 Da, a hydrophilicity index of 1.149, and certain hydrophobicity. The secondary structure was composed of alpha-helix (19 aa, 14.18%), extended strand (48 aa, 35.82%) and random coil (67 aa, 50.00%). The deduced amino acid sequence was used to obtain homologous genes by BLAST, and the phylogenetic tree showed that D. farinae was clustered with D. pteronyssinus. The full-length sequences and expression plasmid of the LepROTL1 gene are obtained, and the molecular features of the gene are demonstrated using bioinformatics analyses, which provide insights into further studies on the gene.

Cloning, expression and immunity analysis of transketolase of Echinococcus granulosus

To obtain the prokaryotic expression of transketolase genes and analyze its value as a diagnostic antigen for echinococcosis. TK gene was amplified by PCR and cloned into prokaryotic vector pMD19-EgTK, and then subcloned into the expression vector pET-28a. The target gene TK prokaryotic expression plasmid pET-28a was constructed and transferred into BL21. The purified protein was identified by SDS-PAGE and Western blotting. The blood samples of patients with cystic echinococcosis (CE group), alveolar echinococcosis (AE group) and healthy people (healthy group) were collected and detected by ELISA with the recombinant EgTK protein as a diagnostic antigen. The recombinant plasmid pET-28a (+)-EgTK was constructed successfully, and there was a band around 70 kDa by using Western blotting. ELISA showed that the difference among the 3 groups of sera reaction A450 was significantly different (F = 44.47, P < 0. 01), and the A450 values ofthe CE group (1.46±0.41) and AE group (1.28±0.29) were higher than that of the healthy group (0.66±0.23), but there was no significant difference between the former two. The recombinant EgTK protein is better to distinguish the echinococcosis group and healthy group, but it can't do a differential diagnosis between CE and AE cases.

Mildew Resistance Locus O Gene Cloning, Characterization, and Expression Pattern in Mulberry (Morus multicaulis) and Its Prokaryotic Expression in E. coli

MLO (mildew resistance locus O), which encodes a transmembrane protein 7TM, is considered to be a model plant gene suitable for studying broad-spectrum resistance. It is a negative regulator of powdery mildew resistance and thus has potential applications in plant breeding. In the present paper, a full cDNA sequence encoding MLO was cloned from the leaves of mulberry (Morus multicaulis) based on mulberry expressed sequence tags (EST), homologous cloning technology, and 5′-RLM-RACE using RT-PCR;the sequence was designated MMLO (GenBank accession no. KX683296). The full cDNA was 1943 bp in length with 5′-untranslated region (UTR) of 106 bp, 3′-UTR of 160 bp, and an open reading frame (ORF) of 1677 bp encoding a protein of 558 amino acids. The estimated molecular weight and isoelectric point (pI) of the putative protein were 62.48 kDa and 9.03, respectively. The MMLO protein had Mlo domain and belonged to the Mlo superfamily. Phylogenetic analysis based on the amino acid sequences encoded by the MLO gene from various species showed that mulberry was closely related to Eucalyptus grandis, Ziziphus jujube, and Juglansregia. Quantitative real-time PCR (qRT-PCR) analysis revealed that MMLO was expressed in all the tissues tested, including leaf, bud, fruit, stem, phloem, and xylem in mulberry with the highest expression in the phloem. The expression level of the mRNA increased and significantly changed under drought, cold, and salt stress treatments compared to the normal growth environment. The ORF segment of the MMLO was inserted into the expression plasmid pET-28a(+) to construct a recombinant expression plasmid. SDS-PAGE result revealed that fusion protein was successfully expressed. Overall, these results provide a better understanding of the molecular basis for the signal transduction mechanism during the stress responses in mulberry trees.

Recombinant laccase from Pediococcus acidilactici CECT 5930 with ability to degrade tyramine.

Biogenic amines degradation by bacterial laccases is little known, so we have cloned and heterologously expressed, in E. coli, a new laccase from Pediococcus acidilactici CECT 5930 (Lpa5930), a lactic acid bacterium commonly found in foods able to degrade tyramine. The recombinant enzyme has been characterized by physical and biochemical assays. Here we report the optimization of expression and purification procedures of this laccase. DNA encoding sequence of laccase from P. acidilactici was amplified by PCR and cloned into the expression plasmid pET28a for induction by isopropyl-β-D-thiogalactoipyranoside. Protein expression was performed in E. coli BL21(DE3) harboring pGro7 plasmid expressing a chaperone folding assistant induced by arabinose. Purification was performed by column metal-chelating chromatography on Ni-NTA-agarose. The laccase enzyme obtained has an apparent molecular mass of ∼60 kDa, an optimum temperature activity toward 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) of 28°C, and was quickly inactivated at temperatures higher than 70°C. The apparent Km value for ABTS was 1.7 mM and the Vmax obtained was 24 U/mg. In addition to ABTS, recombinant Lpa5930 laccase degraded the biogenic amine tyramine at pH 9.5 and pH 4.0 with or without ABTS as a mediator. Tyramine degradation by laccases could solve the problems generated in food due to the presence of this toxic compound.

Characterization and expression patterns of mannose-binding lectin (MMBL) gene in mulberry (Morus multicaulis) and its prokaryotic expression in E. coli

Mannose-binding lectin (MBL) is an important natural immune molecule which plays a significant role in plant self-defense and shows a broad spectrum resistance on plant pathogens, viral pathogens, and fungi. In the present paper, a cDNA sequence encoding MBL, which was designated MMBL (GenBank accession NO: KY348866), was cloned from the leaves of mulberry (Morus multicaulis) based on mulberry expressed sequence tags (EST) and homologous cloning technology using RT-PCR. The cDNA was 978bp in length with a 5′ untranslated regions (UTR) of 189bp, a 3′ UTR of 303bp and an open reading frame (ORF) of 486bp encoding a protein of 161 amino acids. The estimated molecular weight and isoelectric point (pI) of the putative protein were16.97kDa and 6.07, respectively. The MMBL had Jacalin domain and six sugar binding sites, and belonged to Jacalin-like superfamily. Phylogenetic analysis based on the amino acid sequences encoded by the MBL gene from various species showed that mulberry was closely related to Morus notabilis, Ipomoea nil, Capsicum annuum, Solanum lycopersicum and Solanum tuberosum. Quantitative real-time PCR (qRT-PCR) analysis revealed that MMBL was expressed in all the tested tissues, including leaf, bud, fruit, stem, phloem and xylem of the mulberry with the highest expression in the bud and leaf. The expression level of the mRNA has changed significantly under drought, cold, salt and mechanical damage stress treatments compared to the normal growth environment. The ORF segment of the MMBL was inserted into the expression plasmid pET-28a(+) to construct a recombinant expression plasmid. SDS-PAGE and western blot results revealed that His-tagged fusion protein was successfully expressed. Overall, these results showed a better understanding of the molecular basis for the signal transduction mechanism during the stress responses in mulberry trees.

Production of d-psicose from d-glucose by co-expression of d-psicose 3-epimerase and xylose isomerase.

d-Psicose has been drawing increasing attention in recent years because of its medical and health applications. The production of d-psicose from d-glucose requires the co-expression and synergistic action of xylose isomerase and d-psicose 3-epimerase. To co-express these genes, vector pET-28a(+)-dual containing two T7 promoters and RBS sites and an Multiple Cloning Sites was constructed using the Escherichia coli expression plasmid pET-28a(+). The xylose isomerase gene from E. coli MG1665 and the d-psicose 3-epimerase gene from Agrobacterium tumefaciens CGMCC 1.1488 were cloned and co-expressed in E. coli BL21(DE3). After 24h incubation with the dual enzyme system at 40°C, the sugar conversion ratio from d-glucose to d-psicose reached 10%. The optimal conditions were 50°C, pH 7.5 with Co2+ and Mg2+. The d-psicose yields from sugarcane bagasse and microalgae hydrolysate were 1.42 and 1.69g/L, respectively.

Prokaryotic expression of a multi-epitope gene of Xinjiang hemorrhagic fever virus and development of an indirect ELISA for virus detection

Objective To express and purify the multi-epitope peptide of Xinjiang hemorrhagic fever virus (XHFV) in a prokaryotic expression system and to further use it as diagnostic antigen to establish an indirect ELISA for the detection of XHFV in animal sera. Methods A double copy multi-epitope gene (MEPX2) segment with repeated tandem including six highly conservative and dominant B cell epitopes was designed based on the analysis of hydrophilicity and antigenic determinant sites in amino acid sequences of nucleoprotein and glycoprotein from XHFV strain YL04057. The multi-epitope gene MEPX2 was synthesized by chemical method and then inserted into the prokaryotic expression vector pET-32a (+ ) to construct the prokaryotic expression plasmid pET-32a (+ )-MEPX2. The recombinant expression plasmid was transformed into E.coli BL21 (DE3) to induce the expression of rMEPX2 by IPTG. The expressed products were purified by Ni-NTA purification system. The antigenicity of rMEPX2 was identified by Western blot assay. The purified rMEPX2 was used as coating antigen to establish an indirect ELISA by checkerboard for the detection of XHFV in animal serum samples. The results of virus detection were compared with those by using commercial ELISA kit. Results The recombinant expression plasmid pET-32a (+ )-MEPX2 was constructed successfully as confirmed by restriction enzyme digestion and DNA sequencing analysis. Results of the SDS-PAGE and Western blot assay confirmed that the rMEPX2 was expressed correctly with a relative molecular mass (Mr) of about 49×103. The rMEPX2 was recognized specifically by His-tag mouse monoclonal antibody and by positive serum samples of sheep naturally infected with XHFV. The indirect ELISA for the detection of XHFV IgG antibody was established by using the purified rMEPX2 as antigen. Compared with the commercial ELISA kit, the established indirect ELISA showed good specificity for the detection of XHFV in 108 sheep serum samples. Conclusion The recombinant multi-epitope protein rMEPX2 with good antigenicity was obtained successfully. The established indirect ELISA based on the rMEPX2 could be used for the detection of specific antibodies against XHFV. Key words: Xinjiang hemorrhagic fever virus; Multi-epitope peptide; Prokaryotic expression; Indirect ELISA

Recombinant protein of Haemonchus contortus 14-3-3 isoform 2 (rHcftt-2) decreased the production of IL-4 and suppressed the proliferation of goat PBMCs in vitro

14-3-3 proteins have been found to be an excreted/secreted antigen and assumed to be released into the host–parasite interface and described in several unicellular and multicellular parasites. However, little is known about the immunomodulatory effects of H. controtus 14-3-3 protein on host cell. In present study, 14-3-3 isoform 2 gene, designated as Hcftt-2, was amplified by reverse transcription-polymerase chain reaction (RT-PCR) from the adult H. contortus cDNA and cloned into expression plasmid pET32a (+) and expression of the recombinant protein (rHcftt-2) was induced by IPTG. Binding activity of rHcftt-2 to goat peripheral blood mononuclear cells (PBMCs) was confirmed by immunofluorescence assay (IFA) and modulatory effects on cytokine production, cell proliferation, cell migration and nitric oxide (NO) production were observed by co-incubation of rHcftt-2 with goat PBMCs. Sequence analysis showed that it had significant homology with the known 14-3-3 protein isoform 2. Results of IFA revealed that, the rHcftt-2 was bound to the cell surface. We found that, the productions of IL10, IL-17, IFN-γ and cell migration of PBMCs were increased after the cells were incubated with rHCftt-2. However, the productions of IL-4, NO and cell proliferation of the PBMCs were significantly decreased in dose depended manner. Our results showed that the Hcftt-2 played important suppressive regulatory effects on the goat PBMCs.

Effects of missense mutations in sortase A gene on enzyme activity in Streptococcus mutans.

BackgroundStreptococcus mutans (S. mutans) is the major aetiological agent of dental caries, and the transpeptidase Sortase A (SrtA) plays a major role in cariogenicity. The T168G and G470A missense mutations in the srtA gene may be linked to caries susceptibility, as demonstrated in our previous studies. This study aimed to investigate the effects of these missense mutations of the srtA gene on SrtA enzyme activity in S. mutans.MethodsThe point mutated recombinant S.mutans T168G and G470A sortases were expressed in expression plasmid pET32a. S. mutans UA159 sortase coding gene srtA was used as the template for point mutation. Enzymatic activity was assessed by quantifying increases in the fluorescence intensity generated when a substrate Dabcyl-QALPNTGEE-Edans was cleaved by SrtA. The kinetic constants were calculated based on the curve fit for the Michaelis-Menten equation.ResultsSrtA△N40(UA159) and the mutant enzymes, SrtA△N40(D56E) and SrtA△N40(R157H), were expressed and purified. A kinetic analysis showed that the affinity of SrtA△N40(D56E) and SrtA△N40(R157H) remained approximately equal to the affinity of SrtA△N40(UA159), as determined by the Michaelis constant (Km). However, the catalytic rate constant (kcat) and catalytic efficiency (kcat/Km) of SrtA△N40(D56E) were reduced compared with those of SrtA△N40(R157H) and SrtA△N40(UA159), whereas the kcat and kcat/Km values of SrtA△N40(R157H) were slightly lower than those of SrtA△N40(UA159).ConclusionsThe findings of this study indicate that the T168G missense mutation of the srtA gene results in a significant reduction in enzymatic activity compared with S. mutans UA159, suggesting that the T168G missense mutation of the srtA gene may be related to low cariogenicity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12903-016-0204-1) contains supplementary material, which is available to authorized users.

The Analytical and Purification Methodology to Confirm the Presence of a GFP Gene in the Expression Plasmid pET28a Using 6X Histidine-Tag

The Analytical and Purification Methodology to Confirm the Presence of a GFP Gene in the Expression Plasmid pET28a Using 6X Histidine-Tag

Expression, purification of recombinant human cryptochrome I and its application in preparation of protective agent for radiotherapy

Radiotherapy is a treatment for cancer with undesired by-effects. In order to develop a new radiation protective agent that could reduce the by-effects, we tried to express and purify human cryptochrome 1 (hCRY1). The coding sequence of hCRY1 was inserted into prokaryotic expression plasmid pET28a(+), and this protein was purified from Escherichia coli BL21(DE3) after IPTG induction, ultrasonication, inclusion body dissolution, gradient dialysis, nickel column purification and ultrafiltration. The yield of hCRY1 in 1 L E. coli culture (LB medium) was about 10-15 mg. The radiation protective efficiency of hCRY1 was monitored by detecting X-ray-induced H2A.X foci in HaCaT cells. The results of immunofluorescence show that hCRY1 significantly reduces X-ray stimulated DNA damage response. The apoptosis of HaCaT cell was also detected, and the repression of H2A.X foci formation was not due to hCRY1's cytotoxity. All these data suggest a potential application of recombinant hCRY1 as a protective agent for radiotherapy.

Molecular cloning and characterization of a proliferating cell nuclear antigen gene by chemically induced male sterility in wheat (Triticum aestivum L.).

Although a number of studies have shown that chemical hybridizing agents (CHAs) affect anther growth and regulate cell-cycle progression, little is known about the molecular and cellular mechanisms involved. Proliferating cell nuclear antigen (PCNA) is an essential factor in DNA replication, and in many other processes in eukaryotic cells. In this study, the open reading frame of TaPCNA, the PCNA in wheat (Triticum aestivum L.), was cloned by reverse transcription polymerase chain reaction (RT-PCR). Sequence analysis revealed that this gene was 792-bp long and encoded a protein with 234 amino acids. Alignment of the TaPCNA-predicted sequence revealed a high degree of identity with PCNAs from other plant species. A subcellular localization assay indicated that TaPCNA was localized in the nucleus. The TaPCNA was cloned into the prokaryotic expression plasmid pET32a, and the recombinant plasmid was transformed into BL21 (DE3). TaPCNA expression was induced by 0.5 mM isopropyl-beta-D-thiogalactopyranoside and verified using sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blot assays, which indicated that the fusion protein was successfully expressed. The gene involved in the G1-to-S transition, Histone H4, was downregulated by 1376- CIMS, which is a chemically induced male sterility line. However, a semi-quantitative RT-PCR revealed that TaPCNA expression was upregulated in 1376-CIMS. Our results suggest that CHAs (SQ-1) induce DNA damage in wheat anthers. DNA damage results in either the delay or arrest of cell-cycle progression, which affects anther development. This study will help to elucidate the mechanisms of SQ-1-induced male sterility.

Cloning and expression analysis of a peptidoglycan recognition protein in silkworm related to virus infection

In this study, the full-length cDNA of a peptidoglycan recognition protein named BmPGRP-S3 was identified from the silkworm, Bombyx mori by rapid amplification of cDNA ends. It is 807bp and comprises the following: a 5′-untranslated region (UTR) with a length of 112bp, a 3′-UTR with a length of 92bp including a poly-adenylation signal sequence (AATAAA) and a poly(A) tail. The longest open reading frame (ORF) of BmPGRP-S3 is 603bp and encodes a polypeptide of 200 amino acids with a predicted molecular weight of 22.3kDa including a PGRP domain. Sequence similarity and phylogenic analysis results indicated that BmPGRP-S3 belongs to the group of insect PGRPs and is closer to BmPGRP-S4 with the highest identity of 68%. Fluorescent quantitative real-time PCR results revealed that the mRNA transcripts of BmPGRP-S3 were presented in all of the tissues, but were highest in the midgut. In the silkworm larvae infected with B. mori cytoplasmic polyhedrosis virus (BmCPV), the relative expression level of BmPGRP-S3 was upregulated. The DNA segment of a mature BmPGRP-S3 peptide was inserted into the expression plasmid pET-28a(+) to construct a recombinant expression plasmid. Western blot results revealed that mature BmPGRP-S3 could be detected in the hemolymph and midgut which were the most important immune tissues in silkworm. All the results suggested that BmPGRP-S3 may play an important role in the immune response of silkworm to BmCPV infection and provided helpful information for further studying the function of BmPGRP-S3 in silkworm.