Bioinformatics analysis and prokaryotic expression of Strongyloides stercoralis serine protease inhibitor 1 / 中国血吸虫病防治杂志

Objective 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. Methods 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. Results 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. Conclusions The recombinant Ss-SRPN-1 protein has been expressed successfully, and this recombinant protein may be a potential vaccine candidate against strongyloidiasis.

Recombinant expression of Japanese encephalitis virus non-structural protein NS1 gene and its reaction with Flavivirus antigen and antibody / 中国热带医学

@#Abstract： Objective　To elucidate the antigenic antibody reaction of recombinant expression of non-structural protein 1 (NS1) of Japanese encephalitis (JE) virus with various mosquito-borne flaviviruses, including JE virus, and the antigenic antibody reaction of serum samples of patients infected with JE virus in acute stage. Methods　In this study, Escherichia coli prokaryotic expression vector (pET) system was used to recombinant express Japanese encephalitis virus NS1 gene. Western Blot assay was performed to detect the antibody responses of the recombinantly expressed protein against a variety of mosquito-transmitted flaviviruses, including JE virus, as well as antigen-antibody reactions of serum from patients with acute JE virus infection. Results　The NS1 gene expression product of JE virus (P3 strain) was in the form of an inclusion body, and the denatured and renatured expression product was displayed as a single band in the denatured gel (polyacrylamide gel electrophoresis, PAGE), with a molecular weight of about 45 000. The results of further antigen-antibody analysis showed that the antigen/antibody hybridization reaction of the expression product with polyclonal or monoclonal antibody of JE virus (mosquito isolates, encephalitis isolates) and serum samples of patients with acute JE virus infection could be completely consistent. The recombinant product showed negative antigen/antibody hybridization reactions with mosquito-transmitted flaviviruses, such as dengue virus and yellow fever virus polyclonal antibodies, but positive reactions with polyclonal antibodies to West Nile virus and Murray Valley encephalitis virus. Conclusions　In this study, the recombinant expression of the NS1 protein of JE virus was successfully obtained, and the antigen/antibody reaction between the recombinant protein and samples of patients infected with mosquito-borne flavivirus and JE virus was analyzed. The study results provide important basic data for elucidating the antigen-antibody reaction between the NS1 protein of JE virus and mosquito-borne flavivirus. The recombinant expression protein obtained in this study provides an important material basis for further research on the function of JE virus NS1 protein.

Clonagem, expressão recombinante e validação por microarray de proteínas alimentares potencialmente alergênicas / Cloning, recombinant expression and validation by microarray of potentially allergenic food proteins

A maioria das respostas alérgicas a alimentos é mediada por IgE, que pode ser detectada para fins de diagnóstico da alergia alimentar. No entanto, para isso é necessário que alérgenos purificados estejam disponíveis para a elaboração dos diferentes formatos de ensaio, inclusive por microarray, que se constitui em uma ferramenta bastante útil para análise simultânea, e também para a identificação de reatividade cruzada. A esse respeito, é imprescindível ampliar a plataforma de alérgenos que possam ser empregados para a confecção de microarrays. Atualmente, alguns alimentos que constituem objeto de interesse na clínica em função do número de casos de alergia, e sobre os quais as informações a respeito dos alérgenos são escassas, são: abacaxi, mamão, mandioca e manga. Assim, o objetivo desse trabalho foi clonar, expressar e purificar proteínas potencialmente alergênicas de alimentos de importância regional. Após confirmadas por ensaios imunológicos, essas proteínas foram utilizadas na construção e validação de um microarray através de ensaios com os soros de pacientes alérgicos aos alimentos selecionados. Para atingir esse objetivo, foram selecionadas proteínas potencialmente alergênicas coincidentes, apontadas tanto pela similaridade com espécies taxonomicamente mais próximas, quanto pela técnica 2D Western Blotting acoplada à espectrometria de massas. Dezenove proteínas, sendo 4 de abacaxi, 5 de mamão, 6 de mandioca e 4 de manga, foram expressas em Pichia pastoris, purificadas e impressas em um microarray. Após incubar essas proteínas com os soros dos pacientes alérgicos aos alimentos estudados, 18 proteínas mostraram-se potencialmente alergênicas. Além disso, foi observada reatividade cruzada entre proteínas dos alimentos estudados e também em relação ao látex e outros frutos

The majority of allergic reactions to foods is IgE-mediated, which can be detected for the diagnosis of food allergy. However, purified allergens are necessary to produce different kinds of allergy tests, including microarray, which is a useful tool for simultaneous analysis, as well as for the identification of cross-reactivity. In this respect, it is essential to expand the platform of allergens to include them on microarrays. Nowadays, some foods that are object of interest in the clinical area in Brazil and it is necessary a further evaluation about their potential allergens, since there is a limited information about them, are: pineapple, papaya, cassava and mango. Therefore, the aim of this study was cloning, expressing and purifying potentially allergenic proteins of important Brazilian foods. After confirmed by immunological tests, these proteins were used in microarray production and validation by assays with sera from allergic patients to the selected foods. Achieving this goal, matching potentially allergenic proteins were selected, which were identified by comparison among taxonomically closer species (in silico) and 2D Western Blotting coupled with Mass Spectrometry. Nineteen proteins: 4 from pineapple, 5 from papaya, 6 from cassava and 4 from mango were expressed in Pichia pastoris, purified and printed on a microarray. After incubating those proteins with sera from allergic patients to the selected foods, 18 proteins were detected as potentially allergenic. In addition, cross-reactivity was observed among the proteins from the studied foods, and also regarding to the latex and other fruits

Advances in the use of lactic acid bacteria as mucosal delivery vectors of therapeutic molecules / 生物工程学报

Lactic acid bacteria (LAB) are generally recognized as safe food-grade microorganisms and are widely used in food production, preservation, and as probiotics to promote human health. Given the need to develop effective drug delivery strategies, LAB have become attractive live vehicles for the oral, intranasal and vaginal delivery of therapeutic molecules. Being live and safe organisms, LAB are able to directly produce and deliver target proteins for therapeutic purpose, which remarkably reduces the cost for drug production. To date, LAB have been used to deliver a variety of functional proteins to mucosal tissues for the treatment of various diseases. This review summarized the development and application of LAB as mucosal delivery vectors in the last 20 years to provide references for future clinical research.

Effect of E54 mutation of human secreted phospholipase A2 GIIE on substrate selectivity / 生物工程学报

Human secreted phospholipase A2 GIIE (hGIIE) is involved in inflammation and lipid metabolism due to its ability of hydrolyzing phospholipids. To reveal the mechanism of substrate head-group selectivity, we analyzed the effect of mutation of hGIIE on its activity and selectivity. hGIIE structural analysis showed that E54 might be related to its substrate head-group selectivity. According to the sequence alignment, E54 was mutated to alanine, phenylalanine, and lysine. Mutated genes were cloned and expressed in Pichia pastoris X33, and the enzymes with mutations were purified with 90% purity by ion exchange and molecular size exclusion chromatography. The enzymatic activities were determined by isothermal microthermal titration method. The Km of mutant E54K towards 1,2-dihexyl phosphate glycerol decreased by 0.39-fold compared with that of wild type hGIIE (WT), and the Km of E54F towards 1,2-dihexanoyl-sn-glycero-3-phosphocholine increased by 1.93-fold than that of WT. The affinity of mutant proteins with phospholipid substrate was significantly changed, indicating that E54 plays an important role in the substrate head-group selectivity of hGIIE.

Expression of Ietalurus punetaus β-defensin based on recombinant Pichia pastoris / 生物工程学报

β-defensin is a primary protein immune factor in channel catfish's (Ietalurus punetaus) resistance to pathogenic microorganisms. Its primary structure contains a signal peptide composed of 24 amino acid residues at the N-terminal and a mature peptide composed of 43 amino acid residues at the C-terminal. The mature peptide region is responsible for the biological activity of β-defensin. In the present study, a recombinant strain of Pichia pastoris that produces channel catfish β-defensin, was constructed to realize the biosynthesis of channel catfish β-defensin based on eukaryotic expression. First, the β-defensin gene "IPBD" was isolated from the skin of channel catfish by RT-PCR. After linking it with the expression vector pPICZA, pPICZA-IPBD was transferred into competent P. pastoris X-33 cells to obtain recombinant P. pastoris strains. The yeast transformants with multi-copy gene inserts were obtained by using the culture medium containing 1 000 μg/mL zeocin. Using BMM culture medium (without amino nitrogen culture medium) instead of BMMY culture medium (with amino nitrogen culture medium), the fermentation and culture conditions of the recombinant strain were optimized, and the optimal conditions for producing channel catfish β-defensin were determined as follows: the expression was induced for 96 h with 1.0% methanol at 28 °C , 250 r/min. Purified protein with molecular weight of 5.98 kDa was obtained by nickel affinity chromatography, and MALDI-TOF/TOF mass spectrometry proved that it was the expected recombinant IPBD. The antibacterial test results showed that the inhibitory rates of recombinant IPBD on Gram-positive Staphylococcus aureus and Listeria monocytogenes and Gram-negative Pseudomonas aeruginosa were 69.6%, 71.6% and 65.8%, respectively. This study provides a recombinant DNA technique for the development of small molecule natural antibacterial peptide from fish.

Prokaryotic expression, purification and characterization of tissue inhibitor of metalloproteinase-2 / 生物工程学报

Tissue inhibitor of metalloproteinases-2 (TIMP-2) inhibits tumor migration and invasion. Obtaining TIMP-2 protein is conducive to a comprehensive and in-depth study of its function and mechanism in tumorigenesis and development. We collected human TIMP-2 protein through prokaryotic expression in vitro. We expressed, purified and characterized human TIMP-2 protein. First, the human TIMP-2 gene was cloned from the cDNA obtained by reverse transcription of total RNA of human lung cancer A549 cells, and constructed to pET28a vector. The recombinant plasmid pET28a-TIMP-2 was transformed into Escherichia coli BL21(DE3) after restriction endonuclease digestion and sequencing analysis. The expression of TIMP-2 protein was induced by isopropyl-β-D-thiogalactoside (IPTG), and the expression conditions were optimized. After purification by nickel affinity column, the fusion protein His-TIMP-2 was identified by Western blotting method and its biological activity was detected by gelatin zymography. The fusion protein His-TIMP-2 existed in the form of inclusion body in E. coli. In a certain range, the concentration of IPTG had no significant effect on the expression amount of His-TIMP-2. But in this expression system, induction temperature and time were the key parameters, and the expression amount of His-TIMP-2 in E. coli increased with the increase of induction temperature. The purified and refolded fusion protein could effectively inhibit the activity of matrix metalloproteinases expressed by human lung cancer A549 cells. The acquisition of active fusion protein lays a foundation for further study of the function and mechanism of human TIMP-2, and is of great significance for tumor therapy.

Recombinant expression of Crassostrea gigas defensin in Pichia pastoris and its antibacterial activity / 生物工程学报

Defensins are endogenous cationic antimicrobial peptides rich in arginine and cysteine residues. They are important immune factors resisting pathogenic bacteria infection for mollusks. The 43 amino acid residues near the carboxyl terminal for Crassostrea gigas defensin (CgD) form its mature peptide region, responsible for the biological activity of CgD. First, two target genes, CgDH⁺ (with 6×His-tag at 3' end) and CgDH- (without 6×His-tag at 3' end) were separated and amplified by RT-PCR with specific primers from Crassostrea gigas mantle. These two target genes were ligated to the expression vector pPICZαA to construct recombinant expression vectors, pPICZαA-CgDH⁺ and pPICZαA-CgDH-, which were transformed into competent Pichia pastoris X-33 cells by electroporation respectively. The recombinant target proteins, CgDH⁺ and CgDH-, were induced for 72 h with 1% methanol at 29 °C and 250 r/min. The recombinant CgDH⁺ (5.78 kDa) was purified by immobilized metal affinity chromatography (IMAC), and identified by MALDI-TOF-TOF analysis, demonstrating that it was the expected target protein. Based on the concentration of the purified product, the estimated yield of recombinant CgDH⁺ was 2.32 mg/L. Antimicrobial assay showed that the culture medium supernatant containing recombinant CgDH⁺ and recombinant CgDH-, respectively, had activities against Staphylococcus aureus and Pseudomonas aeruginosa, indicating that the existence of 6×His tag in the recombinant proteins do not affect their biological activities.

Expression of the recombinant precursor and putative mature forms of human Interleukin-37 Isoform B (IL-37b) in E. coli expression system

@#<p style="text-align: justify;"><strong>BACKGROUND AND OBJECTIVES: </strong>IL-37b is a cytokine that may exist in several forms, including a full-length precursor protein and its putative mature forms (IL-37b cleaved at E21, 146, and K53, respectively). In recent years, the role of IL-37b has been associated with the regulation of inflammation and inflammatory diseases. Previous studies focused on the intracellular activity of the cytokine, while the bioactivities of its variants when introduced in the extracellular environment has been limited and require further investigation. To enable this, the study produced precursor and truncated forms of IL-37b in an E.coli expression system.</p><p style="text-align: justify;"><strong>METHODOLOGY:</strong> Recombinant proteins of the full-length (FL) and shorter forms (E21, 146, and K53) of IL-37b were produced in IPTG-induced E. coli BL21-CodonPlus(DE3)-RIPL strain and subsequently purified using Ni2+ NTA affinity, ion exchange, and size exclusion chromatography. The identity of the proteins was confirmed through western blotting and LC-MS.</p><p style="text-align: justify;"><strong>RESULTS:</strong> Findings showed that the masses of the expressed proteins correspond to their respective theoretical masses with 24,134.75 +0.04 Da for FL, 21,919.63 +0.80 Da for E21, 19,298.57 +0.04 Da for 146, and 18,551.21 +0.04 Da for K53 at 90-95% purity. This confirms that the correct proteins have been produced and at high purity. Further, the tendency of FL to homodimerize was observed in this study, which may have implications in the extracellular processing and bioactivity of FL.</p><p style="text-align: justify;"><strong>CONCLUSION:</strong> This study describes the successful expression and purification of recombinant precursor and putative mature forms of IL-37b in E.coli, which can be utilized for downstream characterization. </p>

Bioinformatic analysis and characterization of myxobacteria laccase-like multicopper oxidases / 生物工程学报

Laccase is a widely-used environment-friendly copper-containing oxidase found in many plants, insects and fungi. Recently, more and more laccases are also found in bacteria. Myxobacteria are an important bacteria resource. However, myxobacteria are much more difficult to isolate and purify than other bacteria. We used bioinformatic approach to screen myxobacteria proteomes available in NCBI. Based on conserved sequences of four copper binding sites in multicopper oxidase, 30 potential laccase sequences were obtained. Among them, nine genes were synthesized and expressed in Escherichia coli BL21 (DE3). Seven proteins showed laccase activity when tested with traditional laccase substrates. One protein, named rSC-2, was chosen for further research because it exhibited the highest activity towards 2,6-dimethyl phenol (DMP). The molecular weight of rSC-2 was 57 kDa. Its specific activity to DMP was 0.27 U/mg. The optimal temperature and the optimal pH were 60 ℃ and 7.0, respectively. About 50% of the original activity was retained after incubation at 60 ℃ and pH 7.0-8.0 for 1 h. Metals showed different effects on rSC-2. rSC-2 activity was enhanced by several metalsat concentration of 1 mmol/L, such as Ca²⁺ and Mn²⁺. With a higher concentration of 5 mmol/L, the activity of rSC-2 was apparently inhibited. This is the first report of bioinformatics screening myxobacteria laccases in combination with expression in E. coli.

Recombinant expression of hantaan virus protein N with application of Western-blot in detecting anti-hantavirus antibody / 中华流行病学杂志

Objective S gene of hantavirus(HV) was expressed in insect cells by genetic engineering technology.The expression product of S gene was used as antigen to detect anti-HV specific antibody IgG in serum.Methods Gene encoding NP of the strain HV-Z10 was amplified by PCR and then its eukaryotic expression system rBAC-Z 10S-TN was constructed by using the routine genetic engineering method.SDS-PAGE was applied to measure the expression of rNP.Ion-exchange plus Ni-NTA-affinity chromatography was performed to purify the recombinant product.Indirect immuno-fluorescence assay (IFA) was used to determine the specific immune-reactivity of rNP.WB assay was established to detect the serum samples from 95 confirmed HFRS patients.Parameters related to the outcomes of detection were compared with the routine HV-IgG IFA method.Results rBAC-Z10S-TN was able to express rNP with high efficiency.The purified rNP only showed a single protein fragment in the gel after SDS-PAGE.HV IgG could efficiently recognize rNP and hybridize with the recombinant protein.97.67％ of the serum samples from the HFRS patients were positive confirmed by WB.Conclusions We successfully constructed a high efficient prokaryotic expression system of NP encoding gene from hantavirus strain HV-Z10.WB assay which was established in this study could be used as a new serological test for HFRS diagnosis,thanks to the simplicity,safety,sensitivity and specificity of this method.

Recombinant expression of hantaan virus protein N with application of Western-blot in detecting anti-hantavirus antibody / 中华流行病学杂志

Objective S gene of hantavirus(HV) was expressed in insect cells by genetic engineering technology.The expression product of S gene was used as antigen to detect anti-HV specific antibody IgG in serum.Methods Gene encoding NP of the strain HV-Z10 was amplified by PCR and then its eukaryotic expression system rBAC-Z 10S-TN was constructed by using the routine genetic engineering method.SDS-PAGE was applied to measure the expression of rNP.Ion-exchange plus Ni-NTA-affinity chromatography was performed to purify the recombinant product.Indirect immuno-fluorescence assay (IFA) was used to determine the specific immune-reactivity of rNP.WB assay was established to detect the serum samples from 95 confirmed HFRS patients.Parameters related to the outcomes of detection were compared with the routine HV-IgG IFA method.Results rBAC-Z10S-TN was able to express rNP with high efficiency.The purified rNP only showed a single protein fragment in the gel after SDS-PAGE.HV IgG could efficiently recognize rNP and hybridize with the recombinant protein.97.67％ of the serum samples from the HFRS patients were positive confirmed by WB.Conclusions We successfully constructed a high efficient prokaryotic expression system of NP encoding gene from hantavirus strain HV-Z10.WB assay which was established in this study could be used as a new serological test for HFRS diagnosis,thanks to the simplicity,safety,sensitivity and specificity of this method.

Soluble expression of tandem hybrid ubiquitin-binding domains (ThUBD) in prokaryotic cytoplasm of Escherichia coli BL21(DE3) / 军事医学

Objective We increase the soluble expression of artificial tandem hybrid ubiquitin binding domains ( ThUBD) in prokaryotic cytoplasm of Escherichia coli BL21 ( DE3 ) , which offer an effective and special profiling for ubiquitin conjugates( UbC) .Methods Codon optimization of the ThUBD was performed, followed by analysis of codon relative adaptiveness based on relative frequency of synonymous codon ( RFSC) of E.coli.Further induced expression and yeast ubiquitin conjugate enrichment quantified the soluble ThUBD-S and tested the ability to bind UbC.Results The statistical result showed that the percentage of codon of the highest usage frequency was increased from 48%to 75%, and codon adaptation index( CAI) was increased from 0.63 to 0.88 after codon optimization, which might suggest a higher expression of the ThUBD in E.coli BL21 (DE3).The subsequent SDS-PAGE indicated that the soluble target protein was increased four times, which accounted for 13.06%of total cell lysis.Further ubiquitinated proteome of yeast demonstrated that the ability to bind and enrich UbC of optimized ThUBD-S did not change compared with original ThUBD.Conclusion The expression of ThUBD-S can quadruple after codon optimization.At the same time, codon optimization does not impact its soluble expression and the ability to bind UbC.

Expression and purification of Litopenaeus vannamei allergen protein Lit v1.2 / 中国免疫学杂志

Objective:To obtain purified recombinant Litopenaeus vannamei allergen protein Lit v 1.2.Methods: The target gene of Lit v 1.2 was inserted into clone vector pGEM-T and then ligated to the expression vector pET 44a.The pET44a-Liv 1.2 was transformed into Rosetta and screened by ampicillin resistance .The recombinant protein was expressed by IPTG induction .The protein was purified by 6-His tag affinity chromatography and the purification was analyzed by SDS-PAGE gel electrophoresis .Results:The ex-pression plasmid pET44a-Lit v 1.2 was constructed.SDS-PAGE showed that expressed Lit v 1.2 was efficient and soluble in E.coli Rosetta.The protein molecular weight was consistent with the theoretical value .The highly purified target protein was obtained.Conclusion:In this study ,we successfully gained highly purified recombinant allergen protein Lit v 1.2 which was expressed in prokaryotic system and purified by affinity chromatography column .The purified Lit v 1.2 protein will facilitate us to further study its role in immunological responses .

Molecular characteristics, recombinant expression and activity detection of OsGSTL1 from rice

The mRNA of OsGSTL1 was detected in the roots and leaves of rice plants at seedling and tillering stages, and their roots, leaves and panicles at the heading stage. The full-length open reading frame of OsGSTL1 cDNA was 732 bp and encoded a putative polypeptide of 243 amino acids with a calculated molecular mass of 27.30 kDa and a theoretical pI of 5.50. The protein sequences of OsGSTL1 exhibited typical feature of the lambda class GST, which contained the conserved domain "GST_C_Lambda" in C-terminal alpha helical domain and a highly conserved Cys42 in active center. In silico predictions showed that the OsGSTL1 protein was strongly hydrophilic. The phylogenetic analysis revealed OsGSTL1 belonged to monocots subgroup and was closer to IN2-1 of Z. may. The OsGSTL1 gene was cloned into pYTV vector and was introduced into yeast strain PEP4. Western blot analysis showed that the exogenous OsGSTL1 was expressed in the transformed yeast. The GST activity of the crude extracts of yeast showed that the OsGSTL1 transgenic yeast had higher levels of GST activities than the control yeasts. These findings suggested that the OsGSTL1 was a glutathione S-transferase and could play an important role during the growth and development processes in rice.

Distribution of genes encoding LPD of nontypeable Haemophilus influenzae A isolates and the immu-noprotective effects of the expressed recombinant LPD / 中华微生物学和免疫学杂志

Objective To investigate the distribution and sequence conservation of genes encoding the outer membrane lipoprotein D(LPD)of nontypeable Haemophilus influenzae(NTHi)isolates and to ana-lyze the immunogenicity and the immunoprotective effects of the expressed recombinant LPD(rLPD). Meth-ods PCR analysis was used to detect the genes encoding LPD of NTHi isolates. The PCR products were se-quenced after T-A cloning. A prokaryotic expression system for genes encoding LPD was established to ex-press the rLPD. Ni-NTA affinity chromatography was used for purification. SDS-PAGE and Bio-Rad Gel Im-age Analyzer were used to detect the expression and the yield of rLPD. The antigenicity and immunoreactivity of rLPD were detected by ELISA and Western blot assay. The immunoprotective effects of rLPD against lethal dose of NTHi were evaluated in a mouse model. Results All of the tested NTHi isolates were positive for the genes encoding LPD. They shared 98. 0% -99. 4% homologies in nucleotide sequences and 98. 5% -100% homologies in amino acid sequences. The established prokaryotic expression system expressed rLPD with a high yield. High levels of antibody in rabbits were induced by the rLPD. The anti-NTHi antiserum samples from rabbits and children could recognize and react with the rLPD. The result of ELISA indicated that 93. 6%(58 / 62)and 53. 2%(32 / 62)of the serum samples from children with NTHi infection were positive for rLPD-IgM and rLPD-IgG,respectively. The rLPD at concentrations of 100 μg and 200 μg could respectively protect 60. 0% and 73. 3% of mice from lethal NTHi infection. Conclusion The genes enco-ding LPD were extensively distributed in NTHi isolates with high sequence conservation. The expressed rLPD could be used as a potential candidate antigen in the development of genetic engineering vaccine against NTHi infection considering its high immunogenicity and immunoprotective effects.

Cloning and expression of hexon protein of human respiratory adenovirus of three serotypes and analysis of antigenicity of the recombinant proteins / 中华微生物学和免疫学杂志

Objective To clone and express the hexon protein of three prevalent human adenovi -rus strains causing respiratory disease and analyze the antigenic characteristics of the recombinant proteins . Methods The full length genes encoding hexon protein of human adenovirus serotype 3(HAdV3), serotype 4(HAdV4) and serotype 7(HAdV7) were cloned by PCR and sequenced , respectively.The alignment anal-ysis was performed by using hexon gene sequences from GenBank .The major antigenic regions of hexon pro-tein of the three serotypes were expressed in E.coli and purified.The antigenicity, immunogenicity and cross reactivity of the recombinant proteins were determined by ELISA and Western blot assay .Results The full length gene sequence encoding hexon protein of human adenovirus serotype 4 was firstly reported in China , which showed more than 99%homology in both nucleotide and amino acid sequences with the human adeno-virus type 4 NHRC3 strain.The partial hexon protein sequence of HAdV 3, HAdV4 and HAdV7 containing all of the 7 hyperviriable regions ( HVRs) were expressed in E.coli, respectively .The purified recombinant proteins could be recognized by antiserum of the three serotypes of adenovirus .The antiserum samples against the three recombinant proteins could cross-react with particles of the three serotypes of adenovirus . The possible type-and species-specific epitopes were predicted .Conclusion The major antigenic regions of hexon protein of the three serotypes were successfully expressed .The purified recombinant proteins contai-ning both intertypes and type-specific epitopes showed a strong immunogenicity .

Construction of a fusion gene encoding h1a-spaO of Salmonella paratyphi A and analysis of immuno-protective effects of the recombinant protein / 中华微生物学和免疫学杂志

Objective To construct a fusion gene (h1a-spaO) encoding H1a-SpaO protein of Sal-monella paratyphi A ( S.paratyphi A) and to express it in prokaryotic expression system , then to further ana-lyze the immunoprotective effects of the expressed protein rH 1a-SpaO.Methods The h1a-spaO fusion gene formed from separate h1a and spaO genes was amplified by PCR using flexible peptide sequence-containing linking primers and then sequenced after T-A cloning.A prokaryotic expression system for expressing h1a-spaO fusion gene was constructed by using the genetic engineering technique .The expressed protein rH1a-SpaO was examined by SDS-PAGE.The antigenicity and immunoreactivity of rH1a-SpaO protein were deter-mined by Western blot assay .The ability of rH1a-SpaO antiserum agglutinating S.paratyphi A strains was detected by micro-Widal′s test.The immunoprotective effects of rH 1a-SpaO against the lethal dose challenge of S.paratyphi A strains were analyzed in a mouse model and that were compared with those by using equal dose of individual recombinant protein H1a and SpaO (rH1a and rSpaO) as the immunogens, respectively. Results The h1a-spaO fusion gene was 100%identical with the individual h1a or spaO gene in nucleotide and amino acid sequences .The constructed prokaryotic expression system could express the recombinant pro-tein rH1a-SpaO with an advantage of high efficiency .rH1a-SpaO protein was able to react with rH 1a or rSpaO antiserum.Moreover, rH1a-SpaO antiserum also could efficiently recognize rH 1a and rSpaO as well as agglutinate Salmonella paratyphi A strains by binding with H-antigen.The immunoprotective rate (93.3%) in mice pre-immunized with 100 μg of rH1a-SpaO protein was significantly higher than that in those pre-immunized with equal dose of rH1a (60.0%) protein or rSpaO protein(53.3%) (P<0.05).Conclusion The recombinant fusion protein rH 1a-SpaO showed more stronger immunoprotective function than the individ-ual rH1a or rSpaO protein , which could be used as an effective antigen for the development of bi -valent para-typhoid A vaccine or typhoid/paratyphoid capsular polysaccharide-protein combined vaccine .

Gene transfer of Chlorella vulgaris n-3 fatty acid desaturase optimizes the fatty acid composition of human breast cancer cells

Chlorella vulgaris has the gene of n-3 fatty acid desaturase (CvFad3), which can synthesize the precursor of n-3 polyunsaturated fatty acids (PUFAs) or convert n-6 to n-3 PUFAs. The objective of the present study was to examine whether the CvFad3 gene from C. vulgaris can be functionally and efficiently expressed in human breast cancer cells and whether its expression can exert a significant effect on cell fatty acid composition. We inserted the CvFad3 gene into the plasmid pEGFP-C3 to construct the eukaryotic expression vector pEGFP-C3-n-3 and to express the n-3 Fad gene in human breast cancer cells (MCF-7 cells). Transfection of MCF-7 cells with the recombinant vector resulted in a high expression of n-3 fatty acid desaturase. Lipid analysis indicated that the ratio of n-6/n-3 PUFAs was decreased from 6:1 in the control cells to about 1:1 in the cells expressing the n-3 fatty acid desaturase. Accordingly, the CvFad3 gene significantly decreased the ratio of n-6/n-3 PUFAs of the MCF-7 cell membrane. The expression of the CvFad3 gene can decrease cell proliferation and promote cell apoptosis. This study demonstrates that the CvFad3 gene can dramatically balance the ratio of n-6/n-3 PUFAs and may provide an effective approach to the modification of the fatty acid composition of mammalian cells, also providing a basis for potential applications of its transfer in experimental and clinical settings.

Generation of spaO-ompA fusion gene of Salmonella paratyphi A and the immunoprotection of expression product of the fusion gene / 中华微生物学和免疫学杂志

Objective To generate the spaO-ompA fusion gene of Salmonella paratyphi A and its prokaryotic expression system,and to determine the immunoprotection of the recombinant expression product rSpaO-OmpA.Methods A flexible peptide sequence was used to link spaO and ompA genes and a prokaryotic expression system of spaO-ompA fusion gene was subsequently generated.SDS-PAGE and Bio-Rad Agarose Image Analyzer were applied to examine the expression as well as the yield of the target recombinant protein rSpaO-OmpA.The antigenicity and immunoreactivity of rSpaO-OmpA were determined using immunodiffusion test,Western Blot assay and micro-Widal's test.By a mouse infection model,the immunoprotection of rSpaO-OmpA against the lethal challenge of S.paratyphi A was determined.In the animal protective test,the recombinant expressed SpaO (rSpaO) and OmpA ( rOmpA ) were used as the controls.Results The generated spaO-ompA fusion gene had 100％ nucleotide and amino acid sequence identities compared to the single spaO or ompA gene.The constructed prokaryotic expression system IPTG E.coli BL21DE3pET42a-spaO-ompA expressed the recombinant protein rSpaO-OmpA.rSpaO-OmpA combined with the antiserum against wholecell of S.paratyphi A to present positive hybridization signal and induced specific antibody in the immunized rabbits.Immunization with 100 or 200 μg rSpaO-OmpA contributed 66.7％ (8/12) or 83.3％ (10/12) immunoprotective rates in mice when the animals were attacked with S.paratyphi A.The immunoprotective rates produced by rSpaO-OmpA were significantly higher than that of equal rSpaO or rOmpA( P＜0.05 ).The sera from rSpaO-OmpA immunized mice presented 1∶5-1∶40 agglutination titers to the H antigens of different S.paratyphi species,and 1∶1-1∶16 immunodiffusion titers to rSpaO,rOmpA and rSpaO-OmpA proteins,respectively.Conclusion The artificially fusion antigen,rSpaO-OmpA,has more powerful immunogenicity and immunoprotection that the equal rSpaO or rOmpA.