淋球菌外膜蛋白PorB重组质粒的构建及其生物信息学分析与预测
发布时间：2019-05-10 16:48
【摘要】：目的 构建淋病奈瑟菌外膜蛋白PorB (Porin B)的重组质粒pNZ8148-porB，并在宿主菌大肠杆菌MC1061中转化，进一步对其进行生物信息学分析与结构预测，研宄PorB蛋白的理化性质、结构功能域和最佳抗原表位，为后续淋病奈瑟菌疫苗靶位点的研宄奠定基础。 方法 1 pNZ8148-porB重组质粒的构建：根据GeneBank报道的淋球菌PorB基因序列，利用Premier5.0软件设计合成最佳引物，小量提取重组质粒pGEX4T-porB，以pGEX4T-porB为模板，PCR扩增PorB基因，纯化回收后用/? I和顶WIII双酶切PorB基因。将经双酶切的PorB克隆至pNZ8148乳酸杆菌表达载体中，构建表达重组体pNZ8148-porB，，转化大肠杆菌MC1061感受态细胞，然后对重组质粒进行鉴定。 2 PorB蛋白的生物信息学分析与预测：取用本实验室所提供的淋球菌29403标准株的核苷酸序列，运用DNASTAR软件将其核苷酸序列转化成为氨基酸序列，共348个氨基酸残基。应用Expasy—系列在线生物学软件对PorB蛋白的一级、二级结构及表位进行分析与预测。禾II用Protparam、PeptideMass等分析PorB蛋白质的基本理化性质；通过BioEdit或DNASTAR软件、在线工具TMpred等综合分析其疏水区域；利用在线工具TMHMM、Potrer在线工具综合分析其跨膜a-螺旋区、（3-折叠等二级结构信息；ProPred和ProPred-I等进行表位预测分析。 结果 1以淋球菌标准菌株(CMCC29403)基因组DNA和以本实验室保存pGEX4T-porB质粒为模板，PCR成功扩增PorB基因，经酶切、纯化、PCR鉴定分析表明：成功构建了重组体pNZ8148-porB。2以NCBI的MMDB数据库中淋球菌的PIB孔蛋白晶体结构为参考模型，分析预测出PorB蛋白的一级理化性质为稳定性蛋白、二级空间结构为P-折叠型蛋白，亲水疏水性显示为亲水性蛋白，其重要的T淋巴细胞抗原表位为PorB蛋白第77?84、209?217、213?228、278?284、336?342氨基酸序列段，最具潜力的T淋巴细胞抗原表位为外膜区278?284和336?342区段，可能是具有保护性抗原表位的序列。 结论 成功构建重组质粒pNZ8148-porB;经生物信息学分析得出PorB蛋白为理化性质稳定性、（3-折叠型跨膜结构、亲水性蛋白，该蛋白最具疫苗研发潜力的T淋巴细胞抗原优势表位是278?284和336?342氨基酸序列段，为后续PorB蛋白抗原靶位的筛选的研宄及淋病奈瑟菌疫苗的研发提供了参考。
[Abstract]:Objective to construct the recombinant plasmid pNZ8148-porB, of outer membrane protein PorB (Porin B) of Neisseria gonorrhoeae and transform it into host Escherichia coli MC1061. Further bioinformatics analysis and structure prediction were carried out, and the physical and chemical properties of PorB protein were studied. The structural and functional domains and the best antigenic epitopes laid a foundation for the study of the target sites of Neisseria gonorrhoeae vaccine. Methods 1 Construction of pNZ8148-porB recombinant plasmid: according to the PorB gene sequence of Neisseria gonorrhoeae reported by GeneBank, the best primers were designed and synthesized by Premier5.0 software. The recombinant plasmid pGEX4T-porB, was extracted in small quantity and the PorB gene was amplified by PCR. After purification and recovery, use /? The PorB gene was digested with I and top WIII. The PorB was cloned into Lactobacillus pNZ8148 expression vector, and the recombinant pNZ8148-porB, was constructed and transformed into E. coli MC1061 receptive cells, and then the recombinant plasmid was identified. 2 Bioinformatics analysis and prediction of PorB protein: the nucleotides of Neisseria gonorrhoeae 29403 standard strain provided by our laboratory were obtained and transformed into amino acid sequences by DNASTAR software, with a total of 348amino acid residues. The primary, secondary structure and epitope of PorB protein were analyzed and predicted by Expasy- series online biological software. The basic physical and chemical properties of PorB protein were analyzed by Protparam,PeptideMass, and the hydrophobic region of PorB protein was analyzed by BioEdit or DNASTAR software and online tool TMpred. The transmembrane a-helix region, (3-folding and other secondary structure information, ProPred and ProPred-I, etc.) were comprehensively analyzed by using the online tool TMHMM,Potrer online tool. Results 1 using genomic DNA of Neisseria gonorrhoeae standard strain (CMCC29403) and pGEX4T-porB plasmid preserved in our laboratory as templates, PorB gene was successfully amplified by PCR and purified by enzyme digestion. PCR identification analysis showed that the recombinant pNZ8148-porB.2 was successfully constructed with the crystal structure of Neisseria gonorrhoeae PIB pore protein in the MMDB database of NCBI as a reference model, and the primary physical and chemical properties of PorB protein were predicted to be stable proteins. The secondary spatial structure is P-folding protein, hydrophilic hydrophobicity is hydrophilic protein, and its important T lymphocytes antigenic epitope is PorB protein 7784209217213228278284336342amino acid sequence. The most potential antigen epitopes of T lymphocytes are the outer membrane regions 278and 336342, which may be the sequences with protective epitopes. The epitopes of T lymphocytes are the most potential epitopes of T lymphocytes, which may be the sequence of protective epitopes in the outer membrane region. Conclusion the recombinant plasmid pNZ8148-porB; was constructed successfully. The results of bioinformatics analysis showed that PorB protein was physicochemical stability, (3-folding transmembrane structure, hydrophilic protein, the dominant epitope of T lymphocytes antigen which had the most potential for vaccine research and development was the amino acid sequence of 278o284and 336324amino acid). It provides a reference for the subsequent screening of PorB protein antigen target and the development of Neisseria gonorrhoeae vaccine.
【学位授予单位】：大理学院
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R378.16;Q51
本文编号：2473836
[Abstract]:Objective to construct the recombinant plasmid pNZ8148-porB, of outer membrane protein PorB (Porin B) of Neisseria gonorrhoeae and transform it into host Escherichia coli MC1061. Further bioinformatics analysis and structure prediction were carried out, and the physical and chemical properties of PorB protein were studied. The structural and functional domains and the best antigenic epitopes laid a foundation for the study of the target sites of Neisseria gonorrhoeae vaccine. Methods 1 Construction of pNZ8148-porB recombinant plasmid: according to the PorB gene sequence of Neisseria gonorrhoeae reported by GeneBank, the best primers were designed and synthesized by Premier5.0 software. The recombinant plasmid pGEX4T-porB, was extracted in small quantity and the PorB gene was amplified by PCR. After purification and recovery, use /? The PorB gene was digested with I and top WIII. The PorB was cloned into Lactobacillus pNZ8148 expression vector, and the recombinant pNZ8148-porB, was constructed and transformed into E. coli MC1061 receptive cells, and then the recombinant plasmid was identified. 2 Bioinformatics analysis and prediction of PorB protein: the nucleotides of Neisseria gonorrhoeae 29403 standard strain provided by our laboratory were obtained and transformed into amino acid sequences by DNASTAR software, with a total of 348amino acid residues. The primary, secondary structure and epitope of PorB protein were analyzed and predicted by Expasy- series online biological software. The basic physical and chemical properties of PorB protein were analyzed by Protparam,PeptideMass, and the hydrophobic region of PorB protein was analyzed by BioEdit or DNASTAR software and online tool TMpred. The transmembrane a-helix region, (3-folding and other secondary structure information, ProPred and ProPred-I, etc.) were comprehensively analyzed by using the online tool TMHMM,Potrer online tool. Results 1 using genomic DNA of Neisseria gonorrhoeae standard strain (CMCC29403) and pGEX4T-porB plasmid preserved in our laboratory as templates, PorB gene was successfully amplified by PCR and purified by enzyme digestion. PCR identification analysis showed that the recombinant pNZ8148-porB.2 was successfully constructed with the crystal structure of Neisseria gonorrhoeae PIB pore protein in the MMDB database of NCBI as a reference model, and the primary physical and chemical properties of PorB protein were predicted to be stable proteins. The secondary spatial structure is P-folding protein, hydrophilic hydrophobicity is hydrophilic protein, and its important T lymphocytes antigenic epitope is PorB protein 7784209217213228278284336342amino acid sequence. The most potential antigen epitopes of T lymphocytes are the outer membrane regions 278and 336342, which may be the sequences with protective epitopes. The epitopes of T lymphocytes are the most potential epitopes of T lymphocytes, which may be the sequence of protective epitopes in the outer membrane region. Conclusion the recombinant plasmid pNZ8148-porB; was constructed successfully. The results of bioinformatics analysis showed that PorB protein was physicochemical stability, (3-folding transmembrane structure, hydrophilic protein, the dominant epitope of T lymphocytes antigen which had the most potential for vaccine research and development was the amino acid sequence of 278o284and 336324amino acid). It provides a reference for the subsequent screening of PorB protein antigen target and the development of Neisseria gonorrhoeae vaccine.
【学位授予单位】：大理学院
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R378.16;Q51
【参考文献】
相关期刊论文 前1条
1 王彦;张雷;张莉;王涵;;淋病奈瑟菌外膜蛋白PorB基因的克隆及原核表达[J];中华男科学杂志;2011年07期
本文编号：2473836
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