白纹伊蚊唾液腺34 ku-2基因克隆与原核表达

发布时间：2018-10-22 10:33

【摘要】：目的克隆并表达白纹伊蚊唾液腺Aalb_34ku-2蛋白。方法根据白纹伊蚊(罗马株)34ku-2开放读码框序列(Aalb_34ku-2)(GenBank:AY826118.1)设计特异引物,采用RT-PCR技术从白纹伊蚊广州株雌蚊体内扩增获得Aalb_34ku-2基因。利用瑞士生物信息学研究所的蛋白分析专家系统中有关基因和蛋白序列的分析工具,结合其他生物信息学分析软件包,分析预测该蛋白质的结构功能;将该基因克隆到原核表达质粒pET28a(+)中,重组质粒在大肠埃希菌BL21/DE3中经异丙硫代-β-D半乳糖苷(IPTG)诱导表达,表达产物用SDS-PAGE电泳鉴定。结果 RT-PCR扩增获得Aalb_34ku-2 2个转录本。Aalb_34ku-2_1序列长度为954bp,编码318个氨基酸,等电点为5.84;Aalb_34ku-2_2的ORF为882bp,编码294个氨基酸,等电点为6.09。两者相差24个氨基酸;构建Pet28a(+)-Aalb_34ku-2_1重组表达载体并转入大肠埃希菌E.coli BL21(DE3),IPTG诱导后得到的重组蛋白与目的蛋白分子质量(34ku)相符,经鉴定为包涵体蛋白。结论成功构建pET28a(+)-Aalb_34ku-2_1重组原核表达质粒并表达出融合蛋白,为进一步研究该蛋白的功能奠定了基础。
[Abstract]:Objective to clone and express Aalb_34ku-2 protein in salivary gland of Aedes albopictus. Methods according to the 34ku-2 open reading frame (Aalb_34ku-2) sequence (GenBank:AY826118.1) of Aedes albopictus (Rome strain), a specific primer was designed and the Aalb_34ku-2 gene was obtained from female mosquitoes of Guangzhou Aedes albopictus strain by RT-PCR technique. Using the tools of protein analysis expert system of Swiss Institute of Bioinformatics, combining with other bioinformatics analysis software packages, the structure and function of the protein were analyzed and predicted. The gene was cloned into prokaryotic expression plasmid pET28a (). The recombinant plasmid was induced by isoprothio- 尾 -D galactoside (IPTG) in Escherichia coli BL21/DE3. The expression product was identified by SDS-PAGE electrophoresis. Results two transcripts of Aalb_34ku-2 were obtained by RT-PCR amplification. The length of Aalb_34ku-2_1 sequence was 954 BP, encoding 318 amino acids, isoelectric point was 5.84 Aalbu-2-2 ORF was 882bp, encoding 294 amino acids, isoelectric point was 6.09. The recombinant expression vector of Pet28a ()-Aalb_34ku-2_1 was constructed and transformed into Escherichia coli E.coli BL21 (DE3), IPTG). The recombinant protein was identified as inclusion body protein and was consistent with the target protein molecular weight (34ku). Conclusion the recombinant prokaryotic expression plasmid of pET28a ()-Aalb_34ku-2_1 was successfully constructed and the fusion protein was expressed, which laid a foundation for further study of the function of the fusion protein.
【作者单位】：贵阳医学院基础医学院人体寄生虫学教研室;贵阳医学院现代病原生物学实验室;
【基金】：国家自然科学基金项目(No.30600515,81060138) 贵州省省长基金项目(黔省专合字(2007)48号)
【分类号】：R384.1

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