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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。
Objective To clone and express the Aalb_34ku-2 protein of salivary glands of Aedes albopictus. Methods Specific primers were designed according to 34ku-2 open reading frame (Aalb_34ku-2) (GenBank: AY826118.1) of Aedes albopictus (Roman strain), and were amplified by RT-PCR from the female mosquitoes of Aedes albopictus Aalb_34ku-2 gene was obtained. The structure and function of this protein were analyzed and predicted by using the analysis tools of gene and protein sequences in the protein analysis expert system of Swiss Bioinformatics Institute together with other bioinformatics analysis software packages. The gene was cloned into prokaryotic expression vector pET28a (+ ), The recombinant plasmid was induced by IPTG in Escherichia coli BL21 / DE3, and the expressed product was identified by SDS-PAGE electrophoresis. Results Two Aalb_34ku-2 transcripts were obtained by RT-PCR. The sequence length of Aalb_34ku-2_1 was 954bp, encoding 318 amino acids with an isoelectric point of 5.84. The ORF of Aalb_34ku-2_2 was 882bp, encoding 294 amino acids with an isoelectric point of 6.09. The difference between them was 24 amino acids. The recombinant expression vector of Pet28a (+) - Aalb_34ku-2_1 was constructed and transformed into Escherichia coli BL21 (DE3). The recombinant protein induced by IPTG was consistent with the molecular mass of the target protein , Identified as inclusion body protein. Conclusion The recombinant prokaryotic expression plasmid pET28a (+) - Aalb_34ku-2_1 was successfully constructed and the fusion protein was expressed, which laid the foundation for further study on the function of this protein.