【摘要】：目的 構(gòu)建淋病奈瑟菌外膜蛋白PorB (Porin B)的重組質(zhì)粒pNZ8148-porB，并在宿主菌大腸桿菌MC1061中轉(zhuǎn)化，進(jìn)一步對其進(jìn)行生物信息學(xué)分析與結(jié)構(gòu)預(yù)測，研宄PorB蛋白的理化性質(zhì)、結(jié)構(gòu)功能域和最佳抗原表位，為后續(xù)淋病奈瑟菌疫苗靶位點(diǎn)的研宄奠定基礎(chǔ)。 方法 1 pNZ8148-porB重組質(zhì)粒的構(gòu)建：根據(jù)GeneBank報道的淋球菌PorB基因序列，利用Premier5.0軟件設(shè)計合成最佳引物，小量提取重組質(zhì)粒pGEX4T-porB，以pGEX4T-porB為模板，PCR擴(kuò)增PorB基因，純化回收后用/? I和頂WIII雙酶切PorB基因。將經(jīng)雙酶切的PorB克隆至pNZ8148乳酸桿菌表達(dá)載體中，構(gòu)建表達(dá)重組體pNZ8148-porB，，轉(zhuǎn)化大腸桿菌MC1061感受態(tài)細(xì)胞，然后對重組質(zhì)粒進(jìn)行鑒定。 2 PorB蛋白的生物信息學(xué)分析與預(yù)測：取用本實(shí)驗(yàn)室所提供的淋球菌29403標(biāo)準(zhǔn)株的核苷酸序列，運(yùn)用DNASTAR軟件將其核苷酸序列轉(zhuǎn)化成為氨基酸序列，共348個氨基酸殘基。應(yīng)用Expasy—系列在線生物學(xué)軟件對PorB蛋白的一級、二級結(jié)構(gòu)及表位進(jìn)行分析與預(yù)測。禾II用Protparam、PeptideMass等分析PorB蛋白質(zhì)的基本理化性質(zhì)；通過BioEdit或DNASTAR軟件、在線工具TMpred等綜合分析其疏水區(qū)域；利用在線工具TMHMM、Potrer在線工具綜合分析其跨膜a-螺旋區(qū)、（3-折疊等二級結(jié)構(gòu)信息；ProPred和ProPred-I等進(jìn)行表位預(yù)測分析。 結(jié)果 1以淋球菌標(biāo)準(zhǔn)菌株(CMCC29403)基因組DNA和以本實(shí)驗(yàn)室保存pGEX4T-porB質(zhì)粒為模板，PCR成功擴(kuò)增PorB基因，經(jīng)酶切、純化、PCR鑒定分析表明：成功構(gòu)建了重組體pNZ8148-porB。2以NCBI的MMDB數(shù)據(jù)庫中淋球菌的PIB孔蛋白晶體結(jié)構(gòu)為參考模型，分析預(yù)測出PorB蛋白的一級理化性質(zhì)為穩(wěn)定性蛋白、二級空間結(jié)構(gòu)為P-折疊型蛋白，親水疏水性顯示為親水性蛋白，其重要的T淋巴細(xì)胞抗原表位為PorB蛋白第77?84、209?217、213?228、278?284、336?342氨基酸序列段，最具潛力的T淋巴細(xì)胞抗原表位為外膜區(qū)278?284和336?342區(qū)段，可能是具有保護(hù)性抗原表位的序列。 結(jié)論 成功構(gòu)建重組質(zhì)粒pNZ8148-porB;經(jīng)生物信息學(xué)分析得出PorB蛋白為理化性質(zhì)穩(wěn)定性、（3-折疊型跨膜結(jié)構(gòu)、親水性蛋白，該蛋白最具疫苗研發(fā)潛力的T淋巴細(xì)胞抗原優(yōu)勢表位是278?284和336?342氨基酸序列段，為后續(xù)PorB蛋白抗原靶位的篩選的研宄及淋病奈瑟菌疫苗的研發(fā)提供了參考。
[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.
【學(xué)位授予單位】：大理學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R378.16;Q51

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1 王彥;張雷;張莉;王涵;;淋病奈瑟菌外膜蛋白PorB基因的克隆及原核表達(dá)[J];中華男科學(xué)雜志;2011年07期

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