Recombinant Prokaryotic Expression Research Articles

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.

Cloning and catalytic analysis of Isatis indigotica chalcone isomerase in vitro

Chalcone isomerase is a key rate-limiting enzyme in the biosynthesis of flavonoids in higher plants, which determines the production of flavonoids in plants. In this study, RNA was extracted from different parts of Isatis indigotica and reverse-transcribed into cDNA. Specific primers with enzyme restriction sites were designed, and a chalcone isomerase gene was cloned from I. indigotica, named IiCHI. IiCHI was 756 bp in length, containing a complete open reading frame and encoding 251 amino acids. Homology analysis showed that IiCHI was closely related to CHI protein of Arabidopsis thaliana and had typical active sites of chalcone isomerase. Phylogenetic tree analysis showed that IiCHI was classified into type Ⅰ CHI clade. Recombinant prokaryotic expression vector pET28a-IiCHI was constructed and purified to obtain IiCHI recombinant protein. In vitro enzymatic analysis showed that the IiCHI protein could convert naringenin chalcone into naringenin, but could not catalyze the production of liquiritigenin by isoliquiritigenin. The results of real-time quantitative polymerase chain reaction(qPCR) showed that the expression level of IiCHI in the aboveground parts was higher than that in the underground parts and the expression level was the highest in the flowers of the aboveground parts, followed by leaves and stems, and no expression was observed in the roots and rhizomes of the underground parts. This study has confirmed the function of chalcone isomerase in I. indigotica and provided references for the biosynthesis of flavonoid components.

Subunit vaccine based on glycoprotein B protects pattern animal guinea pigs from tissue damage caused by infectious bovine rhinotracheitis virus

Infectious bovine rhinotracheitis (IBR) is caused by Bovine herpesvirus type 1 (BoHV-1), which seriously threatens the global cattle industry. Only vaccination to improve immunity is the most direct and effective means to prevent IBR. Attempts are being made to use subunit vaccines, deleted or recombinant viral vaccines to reduce or eradicate IBR. For investigating the immunological characteristics of glycoprotein B subunit vaccine in pattern animal guinea pigs, the partial glycoprotein B (gB) of BoHV-1 with dominant antigenic characteristic was selected. A recombinant prokaryotic expression vector pET-32a-gB with the truncated gB gene was constructed, expressed, identified and the purified proteins were used to immunize guinea pigs. The immune effect of the subunit vaccine was assessed by monitoring clinical symptoms, viral load, antibody secretion, and histopathological changes. The results indicated that guinea pigs immunized with the gB subunit vaccine produced high levels of anti-gB antibodies and virus-neutralizing antibodies. The gB subunit vaccine significantly reduced viral shedding and lung tissue damage after IBRV challenge. The animals inoculated the gB subunit vaccine also had less virus reactivation. Its protective effect on viral shedding and tissue damage was similar to that of inactivated BoHV-1 vaccine. This work is a proof-of-concept study of subunit vaccine-induced protection against BoHV-1. And it is expected to be a candidate vaccine for the prevention of IBR.

Discovery and confirmation of protein action site AK1 of ginsenosides in brain based on DARTS technology

This study aims to explore the targets of ginsenosides in brain based on drug affinity responsive target stability(DARTS) technology. Specifically, DARTS technology was combined with label-free liquid chromatography tandem mass spectrometry(LC-MS) to screen out the proteins in the brain that might interact with ginsenosides. Based on the screening results, adenylate kinase 1(AK1) was selected for further confirmation. First, the His-AK1 fusion protein was yielded successively through the construction of recombinant prokaryotic expression vector, expression of target protein, and purification of the fusion protein. Biolayer interferometry(BLI) was employed to detect the direct interaction of Rg_1, Re, Rb_1, Rd, Rh_2, F1, Rh_1, compound K(CK), 25-OH-PPD, protopanaxa-diol(PPD), and protopanaxatriol(PPT) with AK1, thereby screening the ginsenoside monomer or sapogenin that had strong direct interaction with the suspected target protein AK1. Then, the BLI was used to further determine the kinetic parameters for the binding of PPD(strongest interaction with AK1) to His-AK1 fusion protein. Finally, molecular docking technology was applied to analyze the binding properties between the two. With DARTS and LC-MS, multiple differential proteins were screened out, and AK1 was selected based on previous research for target verification. Fusion protein His-AK1 was obtained by prokaryotic expression, and the response(nm) of Re, Rg_1, Rd, Rb_1, Rh_1, Rh_2, F1, PPT, PPD, 25-OH-PPD, and CK with His-AK1 was respectively 0.003 1, 0.001 9, 0.042 8, 0.022 2, 0.013 4, 0.037 3, 0.013 9, 0.030 7, 0.140 2, 0.016 0, and 0.040 8. The K_(on), K_(off), and K_D values of PPD and His-AK1 were determined by the BLI as 1.22×10~2 mol~(-1)·L·s~(-1), 1.04×10~(-2) s~(-1), 8.52×10~(-5) mol·L~(-1). According to the molecular docking result, PPD bound to AK1 with the absolute value of the docking score of 3.438, and hydrogen bonds mainly formed between the two. Thus, AK1 is one of the protein action sites of ginsenosides in the brain. The direct interaction between ginsenoside metabolite PPD and AK1 is the strongest.

Apostichopus japonicus matrix metalloproteinase-16 might act as a pattern recognition receptor

Matrix metalloproteinases (MMPs) are an important family of proteinases involved in various physiological processes and associated with the immune response. However, the role of MMPs in the immune response remains unclear. To explore the possible role of MMPs in innate immunity, this study selected the MMP-16 gene encoding peptidoglycan (PGN) binding domain identified in the sea cucumber Apostichopus japonicus (named AjMMP-16, GenBank accession No. AQT26486) for microbial polysaccharide-induced transcriptional expression analysis by quantitative real-time PCR, correlation analysis with nine representative genes from A. japonicus immune pathways in microbial polysaccharide-induced transcriptional expression by using Pearson's correlation test, and prokaryotic recombinant expression. Next, its recombinant protein was employed for microbial polysaccharide-binding analysis with ELISA and bacterial binding analysis with the indirect immunofluorescence method. The results showed that AjMMP-16 was significantly induced by diaminopimelic acid (DAP)-type PGN, lipopolysaccharide, mannan, and β-1,3-glucan and was closely correlated with myeloid differentiation factor 88 (MyD88) in microbial polysaccharide-induced transcriptional expression. In addition, recombinant AjMMP-16 bound to lysine-type PGN, DAP-type PGN, lipopolysaccharide, mannan, β-1,3-glucan, Vibrio splendidus, Pseudoalteromonas nigrifaciens, Shewanella baltica, Bacillus cereus, Escherichia coli, and Staphylococcus aureus. These results suggest that AjMMP-16 might act as a pattern recognition receptor in innate immunity and play an important role in initiating the MyD88-dependent Toll-like receptor signaling pathway.

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.

Identification and immunogenicity of NFA47650 protein from Nocardia farcinica

Objective To construct the prokaryotic expression vector of NFA47650 protein from Nocardia farcinica, induce the expression and purification of NFA47650 protein, and analyze its immunogenicity. Methods The primers were designed and synthesized based on nfa-47650 gene sequence of N. farcinica; pET30a (+) expression vector was constructed by PCR amplification; NFA47650 protein was purified after expressing in E. coli BL21. The subcellular localization and immunogenicity with NFA47650 mice anti-serum were analyzed by Western Blot. Results The prokaryotic recombinant expression vector of N. farcinica NFA47650 protein was successfully constructed, which was efficiently expressed in BL21 E. coli as inclusion bodies. Western Blot showed that NFA47650 protein was mainly located in the culture supernatant. In addition, this protein could not only be specifically identified by the antisera from mice and rabbits infected with N. farcinica, but also cross-reacted with antisera from mice infected with N. brasiliensis and N. cyriacigeorgica. Conclusion The NFA47650 protein is an important secreted protein, which can trigger immune response by reacting with the antisera of different Nocardia species.

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.

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。

Heterologous and Homologous Expression of Proteins from Haloarchaea: Denitrification as Case of Study.

Haloarchaea (halophilic microbes belonging to the Archaea domain) are microorganisms requiring mid or even high salt concentrations to be alive. The molecular machinery of these organisms is adapted to such conditions, which are stressful for most life forms. Among their molecular adaptations, halophilic proteins are characterized by their high content of acidic amino acids (Aspartate (Asp) and glumate (Glu)), being only stable in solutions containing high salt concentration (between 1 and 4 M total salt concentration). Recent knowledge about haloarchaeal peptides, proteins, and enzymes have revealed that many haloarchaeal species produce proteins of interest due to their potential applications in biotechnology-based industries. Although proteins of interest are usually overproduced in recombinant prokaryotic or eukaryotic expression systems, these procedures do not accurately work for halophilic proteins, mainly if such proteins contain metallocofactors in their structures. This work summarizes the main challenges of heterologous and homologous expression of enzymes from haloarchaea, paying special attention to the metalloenzymes involved in the pathway of denitrification (anaerobic reduction of nitrate to dinitrogen), a pathway with significant implications in wastewater treatment, climate change, and biosensor design.

Effects of host fatty acid-binding protein 4 on Eimeria tenella sporozoites invasion of cells.

In our previous study, proteomics analyses of host cells infected with Eimeria tenella sporozoites coupled with isobaric tags for relative and absolute quantitation, identified several host proteins related to Eimeria invasion. In this study, A 458-bp Gallus gallus fatty acid-binding protein 4 (FABP4) gene was cloned and subcloned to pET-28c(+) vector to construct the prokaryotic recombinant expression plasmid pET-28c(+)-FABP4. The 18.5kDa recombinant FABP4 protein (rFABP4) was expressed and identified by western blotting. Expression of FABP4 in E. tenella sporozoite-infected DF-1 cells was downregulated significantly than in non-infected cells detected by western blotting and immunohistochemistry. The antibody inhibition assay showed that antibodies against FABP4 at 50, 100, 200, 300, and 400μg/mL had no significant effect on sporozoite invasion. BMS-309403 and transforming growth factor-β3 (TGF-β3) was used to inhibit and improve the expression of FABP4 in DF-1 cells, respectively, and their effect on the sporozoite invasion of cells was detected by flow cytometry. Sporozoite invasion rate in the BMS-309403-treated group was not significantly affected; however, the invasion rate in the TGF-β3-treated group declined significantly. These results show that host FABP4 plays a negative role in Eimeria invasion. However, further studies are needed to elucidate the exact mechanism of how FABP4 negatively regulates Eimeria invasion.

Genome-wide screening of hexokinase gene family and functional elucidation of HXK2 response to cold stress in Jatropha curcas.

Hexokinase, the key rate-limiting enzyme of plant respiration and glycolysis metabolism, has been found to play a vital role in plant sugar sensing and sugar signal transduction. Using Jatropha curcas genome database and bioinformatics method, J. curcas HXK gene family (JcHXK) was identified and its phylogenetic evolution, functional domain, signal peptide at the N-terminal, and expression analysis were conducted. The results showed that a total of 4 HXK genes (JcHXK1, JcHXK2, JcHXK3, and JcHKL1) with 9 exons were systematically identified from J. curcas. JcHXK1, JcHXK3, and JcHKL1 with putative transmembrane domain at the N-terminal belonged to the type of secretory pathway protein, and JcHXK2 contained putative chloroplast targeting peptide. Quantitative real-time PCR (qRT-PCR) analysis revealed that all the four JcHXKs were expressed in different tissues of the leaves, roots, and seeds; however, JcHXK1 and JcHKL1 expression were higher in the roots, whereas JcHXK2 and JcHXK3 showed over-expression in the leaves and seeds, respectively. Furthermore, all the four JcHXKs were up-regulated in the leaves after cold stress at 12°C; however, only JcHXK3 remarkably demonstrated cold-induced expression in the roots, which reached the highest expression level at 12h (2.28-fold). According to the cis-acting element analysis results, JcHXK2 contained the most low temperature responsive elements, which was closely related to the cold resistance in J. curcas. A pET-28a-JcHXK2 prokaryotic recombinant expression vector was successfully constructed and a 57.0kDa protein was obtained, JcHXK2 revealed catalytic activity towards glucose and fructose, with a higher affinity for glucose than fructose. The subcellular localization assays revealed that JcHXK2 was localized in the chloroplast. The results of this study might provide theoretical foundation for further studies on gene cloning and functional verification of HXK family in J. curcas.

Cloning, prokaryotic expression and function of the Bufo bufo gargarizans 3β-hydroxysteroid dehydrogenase (3βHSD) gene

Bufadienolides, one kind of steroids, are the major active component secreted by ear-side gland of Bufo species. Preliminary studies on high-throughput transcriptome sequencing about B. bufo gargarizans showed that the expression of 3β-Hydroxysteroid dehydrogenase (3βHSD) in ear-side gland was nearly 20 times higher than that in liver. The enzyme 3βHSD is an essential step in the biosynthesis of steroid such as progesterone, estrogens and androgens in steroidogenic tissues. Accordingly, 3βHSD is probably an important enzyme involved in the biosynthesis of bufadienolides. In this study, Bbg-3βHSD cDNA was cloned from the ear-side gland of B. bufo gargarizans. Genetic engineering techniques were used to construct a recombinant prokaryotic fusion expression plasmid pCOLD-Bbg3βHSD which was introduced into E. coli BL21 for prokaryotic expression. Bbg-3βHSD has an open reading frame (ORF) of 1134 bp and encodes 377 amino acid residues. The speculated protein molecular weight is 42.8 kDa and its theoretical isoelectric point is 8.68. Amino acid sequence homologous analysis showed that Bbg-3βHSD was highly homologous to the 3βHSD protein of other species. Phylogenetic tree showed the highest similarity between Bbg-3βHSD and 3βHSD from Rana rugosa. The optimized expression of recombinant Bbg-3βHSD were achieved by inducing with 0.1 mmol L−1 IPTG at 15 °C for 20 h. Enzymatic activity in vitro shows that pregnenolone and dehydroepiandroesterone could be 3β-oxidized by Bbg-3βHSD when NAD+ was used as the coenzyme. Enzymatic properties showed that the optimum reaction temperature of recombinant Bbg-3βHSD was 40 °C, the optimum pH was 8.5, and the optimum coenzyme concentration was 1.5 mmol L−1.

Gene cloning, expression and immune adjuvant properties of the recombinant fusion peptide Tα1-BLP on avian influenza inactivate virus vaccine

Thymosin α1 (Tα1) and bursin-like peptide (BLP) are both immunopotentiators. In order to investigate adjuvant of thymosin α1-bursin-like peptide (Tα1-BLP), we cloned the gene of Tα1-BLP and provided evidence that the gene of Tα1-BLP in a recombinant prokaryotic expression plasmid was successfully expressed in E. coli BL21. To evaluate the immune adjuvant properties of Tα1-BLP, chickens were immunized with Tα1-BLP combined with H9N2 avian influenza whole-inactivated virus (WIV). The titers of HI antibody, antigen-specific antibodies, AIV-neutralizing antibodies, levels of Th1-type cytokines (IFN-γ) and Th2-type cytokines (IL-4) and lymphocyte proliferation responses were determined. What's more, the viral loads and pathologic changes of lung tissue were observed by virus challenge experiment and HE staining to evaluate the immune protection of chickens. We found that Tα1-BLP enhanced HI antibody and antigen-specific IgG antibodies titers, increased the level of AIV-neutralizing antibodies, induced the secretion of Th1- and Th2-type cytokines, and promoted the proliferation of T and B lymphocyte, Furthermore, virus challenge experiment and HE staining confirmed that Tα1-BLP contributed to inhibition replication of the virus from chicken lungs and protected the lungs from damage. Altogether, this study suggested that Tα1-BLP is a novel adjuvant suitable for H9N2 avian influenza vaccine.

Cloning and characterization of two glutathione S-transferases cDNAs in Foc4 and expression under exogenous oxidative stress

In order to identify two putative glutathione S-transferase (GSTs) genes in Fusarium oxysporum f. sp. cubense race 4 (Foc4), cDNA sequences of the entire coding regions of the two genes were cloned from Foc4 using RT-PCR method. Subsequently, the two genes were named Fogst1 and Fogst2 respectively. The length of open reading frame of Fogst1 was 609 bp and encoded a protein including 202 amino acid residues, Fogst2 possessed an open reading frame with 693 bp which encoded a 230-amino acid protein. Phylogenetic analysis showed that Fogst1 belonged to sigma (σ) subtype members of the GSTs superfamily, and Fogst2 was a new member of an unknown subfamily in the GSTs superfamily. To verify the expression of Fogst1 and Fogst2, the recombinant prokaryotic expression vector pET28a-Fogst1 and pET28a-Fogst2 were constructed and transformed into Escherichia coli expression strain BL21(DE3). The soluble recombinant proteins Fogst1 and Fogst2 were obtained after being induced by IPTG. GSTs activity assays showed that both of the two recombinant proteins had specific activity with CDNB. For real time RT- PCR analysis, the mycelium samples of Foc4 were collected after treatment by H₂O₂ for 1, 5, 12, 24 hours. The results showed that the expression of Fogst1 and Fogst2 were significantly up-regulated in the first 5 hours, and then decreased and returned to normal level. These results suggested that Fogst1 and Fogst2 may be involved in the process of Foc4 resistance to exogenous oxidative stress.

Design and immunogenicity evaluation for the bacteria-like particle vaccine against swine type O foot-and-mouth disease virus

Based on gram positive enhancer matrix displaying technology, we designed and evaluated a bacteria-like particle vaccine against swine type O Foot-and-mouth disease virus. Three optimized genes of type O Foot-and-mouth disease virus strain Mya98 were cloned into recombinant prokaryotic expression vector pQZ-PA and renamed as pQZ-BT1B-PA, pQZ-BT2B-PA and pQZ-B (T1BT2) 4B-PA, fused with an anchor protein (PA) binding to Gram-positive enhancer matrix (GEM) particles specifically. The protein expression was identified with SDS-PAGE and Western blotting, and then purified with GEM particles. Five-week old female mice were randomly divided into six groups and all the immunization was developed according to subcutaneous injection. Mice in the first three groups were injected with 50 μg/dose GEM-BT1B, GEM-BT2B and GEM-B (T1BT2) 4B, respectively. Mice in the fourth group were immunized with commercial peptide vaccine as positive control. The fifth group vaccinated with host E. coli transformed with pQZ-PA fulfilled as negative control. Mice in the last group injected with sterile PBS served as blank control. The humoral immunity of recombinant protein vaccine was evaluated with peptide-specific antibody and LPB antibody. The cellular immunity was evaluated with lymphocyte proliferation test and cytokine expression detection. SDS-PAGE and Western blotting showed that the most part of soluble target fusion protein have been purified and displayed on GEM particles. Vaccine GEM-B (T1BT2) 4B stimulated mice produce not only higher level of specific antibody against peptide and Foot-and-mouth disease virus specific liquid phase blocking antibody, but also more vigorous spleen lymph proliferation and higher levels of Th1 type cytokines. To summarize, vaccine of GEM-B (T1BT2) 4B possessed good immunogenicity and opened a new way for further Foot-and-mouth disease virus subunit vaccine design.

Polymorphic membrane protein I of Chlamydia trachomatis: prokaryotic expression, purification, antibody preparation and identification

Objective To clone and express the polymorphic membrane protein I (PmpI) gene of Chlamydia trachomatis (Ct) , and to assess the immunogenicity and biological characteristics of PmpI. Methods A bioinformatic software was used to analyze the sequence of the PmpI gene of Ct, and to predict B cell epitopes in PmpI. With Ct serovar D DNA as the template, PCR was performed to amplify the N-terminal region (from position 90 to 1464) of the PmpI gene, which was cloned into a prokaryotic expression vector pET28a to express the recombinant protein PmpI. A Ni-ion affinity chromatography column was used to purify the recombinant protein, which was used to immunize New Zealand rabbits for preparation of polyclonal antibodies. Western blot analysis was conducted to evaluate the immunogenicity of this protein. Results A comprehensive analysis was carried out on the secondary structure, flexible regions, hydrophilicity plot, antigenic index and surface probability plot of the protein, which suggested that PmpI had 8 dominant B-cell epitopes. The product of PCR targeting the PmpI gene of Ct serovar D showed a total length of 1 375 bp. The recombinant prokaryotic expression vector pET28a-PmpI was successfully constructed. A recombinant protein with a relative molecular mass of approximately 50 000 was successfully expressed after isopropyl β-d-1-thiogalactopyranoside (IPTG) induction, and purified by affinity chromatography. Polyclonal antibodies against the recombinant protein were successfully prepared. Conclusion The N-PmpI protein of Ct serovar D is cloned and expressed successfully, laying a foundation for further studies on its biological functions. Key words: Chlamydia trachomatis; Bacterial outer membrane proteins; Cloning, molecular; Polymorphic membrane protein

Molecular Cloning and Expression Analysis of a Hexokinase Gene, MdHXK1 in Apple

A hexokinase gene named MdHXK1 (MDP0000309677) was cloned from ‘Gala’ apple (Malus × domestica Borkh.). Sequence analysis showed that the MdHXK1 gene was 1 497 bp long and encoded 499 amino acids. The predicted molecular mass of this protein was 54.05 kD, and the pI was 5.76. A phylogenetic tree indicated apple MdHXK1 exhibited the highest sequence similarity to Pyrus bretschneideri PbHXK1. Analysis of the functional domain showed that the MdHXK1 protein included two conserved kinase domains. The prediction of subcellular localization suggested that the MdHXK1 protein was mainly localized in the cytoplasm. There was an indication that MdHXK1 existed as one copy in the apple genome by Southern blotting. Silico analysis suggested that the promoter sequence contained several typical cis-acting elements, including defense, sugar signaling and phytohormone responsive elements. Quantitative real-time PCR analysis demonstrated that the MdHXK1 gene was mainly expressed in stem and flower tissues. During the development of apple fruits, the expression of the MdHXK1 gene initially increased and then decreased. The changes on Glc phosphorylation relative activity and glucose concentration showed the same trend. In addition, the expression of this gene was induced by salt stress, low temperature, and abscisic acid (ABA). Finally, we obtained and purified the fused MdHXK1 protein by recombinant prokaryotic expression. Studies have demonstrated that MdHXK1 may participate in sugar metabolism in apple fruits. Enzyme encoded by MdHXK1 is a key factor in the mediation of sugar accumulation. Recently, researchers on hexokinase at home and abroad mainly focused on model plants, such as Arabidopsis, tobacco and rice, but orchard fruit like apple were underresearched. Our research established the foundation for the further study of the functions of MdHXK1.

The recombinant expression and activity detection of MAF-1 fusion protein.

This study establishes the recombinant expression system of MAF-1 (Musca domestica antifungal peptide-1) and demonstrates the antifungal activity of the expression product and shows the relationship between biological activity and structure. The gene segments on mature peptide part of MAF-1 were cloned, based on the primers designed according to the cDNA sequence of MAF-1. We constructed the recombinant prokaryotic expression plasmid using prokaryotic expression vector (pET-28a(+)) and converted it to the competent cell of BL21(DE3) to gain recombinant MAF-1 fusion protein with His tag sequence through purifying affinity chromatographic column of Ni-NTA. To conduct the Western Blotting test, recombinant MAF-1 fusion protein was used to produce the polyclonal antibody of rat. The antifungal activity of the expression product was detected using Candida albicans (ATCC10231) as the indicator. The MAF-1 recombinant fusion protein was purified to exhibit obvious antifungal activity, which lays the foundation for the further study of MAF-1 biological activity, the relationship between structure and function, as well as control of gene expression.

EST-based in silico identification and in vitro test of antimicrobial peptides in Brassica napus

BackgroundBrassica napus is the third leading source of vegetable oil in the world after soybean and oil palm. The accumulation of gene sequences, especially expressed sequence tags (ESTs) from plant cDNA libraries, has provided a rich resource for genes discovery including potential antimicrobial peptides (AMPs). In this study, we used ESTs including those generated from B. napus cDNA libraries of seeds, pathogen-challenged leaves and deposited in the public databases, as a model, to perform in silico identification and consequently in vitro confirmation of putative AMP activities through a highly efficient system of recombinant AMP prokaryotic expression.ResultsIn total, 35,788 were generated from cDNA libraries of pathogen-challenged leaves and 187,272 ESTs from seeds of B. napus, and the 644,998 ESTs of B. napus were downloaded from the EST database of PlantGDB. They formed 201,200 unigenes. First, all the known AMPs from the AMP databank (APD2 database) were individually queried against all the unigenes using the BLASTX program. A total of 972 unigenes that matched the 27 known AMP sequences in APD2 database were extracted and annotated using Blast2GO program. Among these unigenes, 237 unigenes from B. napus pathogen-challenged leaves had the highest ratio (1.15 %) in this unigene dataset, which is 13 times that of the unigene datasets of B. napus seeds (0.09 %) and 2.3 times that of the public EST dataset. About 87 % of each EST library was lipid-transfer protein (LTP) (32 % of total unigenes), defensin, histone, endochitinase, and gibberellin-regulated proteins. The most abundant unigenes in the leaf library were endochitinase and defensin, and LTP and histone in the pub EST library. After masking of the repeat sequence, 606 peptides that were orthologous matched to different AMP families were found. The phylogeny and conserved structural motifs of seven AMPs families were also analysed. To investigate the antimicrobial activities of the predicted peptides, 31 potential AMP genes belonging to different AMP families were selected to test their antimicrobial activities after bioinformatics identification. The AMP genes were all optimized according to Escherichia coli codon usage and synthetized through one-step polymerase chain reaction method. The results showed that 28 recombinant AMPs displayed expected antimicrobial activities against E. coli and Micrococcus luteus and Sclerotinia sclerotiorum strains.ConclusionThe study not only significantly expanded the number of known/predicted peptides, but also contributed to long-term plant genetic improvement for increased resistance to diverse pathogens of B.napus. These results proved that the high-throughput method developed that combined an in silico procedure with a recombinant AMP prokaryotic expression system is considerably efficient for identification of new AMPs from genome or EST sequence databases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1849-x) contains supplementary material, which is available to authorized users.