致倦库蚊天蚕素（Cecropin）基因家族的克隆和表达分析

发布时间：2018-04-19 01:14

本文选题：致倦库蚊 + 天蚕素基因家族　；参考：《贵州医科大学》2016年硕士论文

【摘要】：目的:克隆致倦库蚊Cecropin基因家族的4个基因;比较分析Cecropin家族基因的结构、氨基酸序列、蛋白理化特性,预测蛋白空间结构,以及其进化关系;了解Cecropin家族基因在致倦库蚊不同发育时期的表达谱;选择Cec B2进行原核表达,检测Cec B2多肽的抑菌活性。方法:通过生物信息学方法从致倦库蚊基因组和转录组中筛选出致倦库蚊Cecropin基因家族的4个基因,并进行序列分析和系统发育分析;采用RT-PCR方法从致倦库蚊c DNA中扩增出Cecropin家族4个基因,与p MD-18T载体连接后测序鉴定;提取致倦库蚊不同发育时期的总RNA,通过半定量PCR检测4个Cecropin基因在不同龄期的表达;构建Cec B2重组表达质粒,鉴定正确后转入Rosetta表达菌中,优化IPTG诱导浓度和诱导时间,表达纯化重组蛋白,并采用SDS-PAGE和Western blot对重组蛋白进行检测和鉴定,进一步透析、浓缩及肠激酶切割纯化的融合蛋白,采用Tricine-SDS-PAGE检测Cec B2目的蛋白。同时通过化学合成方法合成足量Cec B2成熟肽,并进行体外抑菌活性检测。结果:1.致倦库蚊Cecropin基因家族的编码氨基酸平均长度约60aa,编码蛋白的平均理论分子量和平均等电点p I值分别为6389.0和10.69,为小分子碱性蛋白;系统进化分析表明致倦库蚊Cecropin与冈比亚按蚊和埃及伊蚊亲缘关系较近。2.半定量PCR研究发现,Cec B2在整个发育时期均有表达,而Cec B1,Cec A1,Cec A2除卵期无明显表达外其余时期均有不同程度表达,总的表达趋势为随虫龄的增长而增加。3.成功构建原核表达载体p ET32a-Cec B2,最佳表达条件为:0.05mmol/L IPTG诱导3h,表达纯化获得大小约25k D的融合蛋白,Western blot鉴定融合蛋白可与鼠抗His-tag单克隆抗体发生抗原抗体结合反应,证明成功获得Cec B2融合蛋白,但本研究获得融合蛋白不具有抑菌活性。4.化学合成Cec B2多肽分别对G+菌(金黄色葡萄球菌、白色葡萄球菌、溶壁微球菌),G-菌(大肠埃希菌)以及真菌(白色念珠菌)均表现出不同程度的抑菌活性,细菌最大抑菌圈达到17mm(溶壁微球菌),真菌抑菌圈达到8mm(白色念珠菌)。结论:1.致倦库蚊Cecropin为小分子碱性蛋白。2.Cec B2在整个发育时期均有表达,Cec B1,Cec A1,Cec A2除卵期无明显表达外其余时期均有不同程度表达。3.成功构建p ET32a-Cec B2原核表达载体,获取了大小约25k D的可溶性融合蛋白。4.Cec B2合成多肽对G+菌,G-菌以及真菌(白色念珠菌)具有不同程度抑菌作用,推测其具有潜在的抗生素研发意义。
[Abstract]:Objective: to clone four genes of Cecropin gene family of Culex pipiens quinquefasciatus, compare and analyze the structure, amino acid sequence, physicochemical properties of protein, predict the spatial structure of protein and its evolutionary relationship.To investigate the expression profiles of Cecropin family genes in different developmental stages of Culex pipiens quinquefasciatus, Cec B2 was selected for prokaryotic expression and the bacteriostatic activity of Cec B2 polypeptide was detected.Methods: four genes of Cecropin gene family of Culex pipiens quinquefasciatus were screened by bioinformatics from the genome and transcriptome of Culex pipiens quinquefasciatus, and sequenced and phylogenetic analysis were performed.Four Cecropin family genes were amplified from c DNA of Culex pipiens quinquefasciatus by RT-PCR method and sequenced with p MD-18T vector. The total RNAs of Culex quinquefasciatus at different developmental stages were extracted, and the expression of four Cecropin genes at different ages were detected by semi-quantitative PCR.The recombinant expression plasmid of Cec B2 was constructed, identified correctly and then transferred into Rosetta expression strain. The recombinant protein was expressed and purified by optimizing the concentration and time of IPTG induction. The recombinant protein was detected and identified by SDS-PAGE and Western blot for further dialysis.The fusion protein was purified by concentration and enterokinase cleavage. Cec B2 target protein was detected by Tricine-SDS-PAGE.At the same time, the mature peptide of Cec B2 was synthesized by chemical synthesis method, and the antibacterial activity was tested in vitro.The result is 1: 1.The average length of encoded amino acids in the Cecropin gene family of Culex pipiens quinquefasciatus was about 60aa, and the average theoretical molecular weight and average isoelectric point (Pi) of the encoded proteins were 6389.0 and 10.69, respectively.Phylogenetic analysis showed that Cecropin was closely related to Anopheles gambiae and Aedes aegypti.The results of semi-quantitative PCR showed that Cec B2 was expressed during the whole development period, while Cec B1Cec A1CA2 was expressed in different degrees at all stages except in egg stage, and the total expression trend was increased with the increase of worm age.The prokaryotic expression vector p ET32a-Cec B2 was successfully constructed. The optimal expression conditions were as follows: 0. 05 mmol / L IPTG was induced for 3 h. The fusion protein was purified and purified by Western blot. The fusion protein was identified to react with mouse anti His-tag monoclonal antibody.It was proved that the fusion protein of Cec B2 was successfully obtained, but the fusion protein had no bacteriostatic activity. 4.The chemically synthesized Cec B2 peptides showed different degrees of bacteriostatic activity against G bacteria (Staphylococcus aureus, Staphylococcus albicans, Micrococcus micrococci G- (Escherichia coli) and fungi (Candida albicans), respectively.The maximum bacteriostasis of bacteria was 17mm (micrococcus lysoid, 8mm) (Candida albicans).Conclusion 1.The Cecropin of Culex pipiens quinquefasciatus was a small molecular basic protein. 2. Cec B2 was expressed in different degrees during the whole development period except for the egg stage.The prokaryotic expression vector of p ET32a-Cec B2 was successfully constructed, and the soluble fusion protein of about 25kD was obtained. 4. The synthetic peptide of Cec B2 had different degree of bacteriostatic effect on G- and fungi (Candida albicans).It is speculated that it has potential significance in antibiotic research and development.
【学位授予单位】：贵州医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q78

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