白纹伊蚊唾液腺34 ku-2基因克隆与原核表达

Cloning and prokaryotic expression of the gene coding for salivary secreted protein 34ku-2 from Aedesalbopictus

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-2protein of Aedes albopictus. Methods The ORF of Aalb_34ku-2from the Guangzhou strain of Ae.albopictus was cloned with gene-specific primers designed based on Aalb_34ku-2from the Rome strain of Ae.albopictusin GenBank（No.AY826118.1）.The sequence was analyzed using relevant tools available at ExPaSy,the bioinformatics portal of the Swiss Institute of Bioinformatics,and the other bioinformatic software.The sequence of Aalb_34ku-2encoding a mature peptide was further cloned into a prokaryotic expression vector,PET28a（＋）.The recombinant plasmids were transformed into E.coli BL21/DE3 and expressed by induction with IPTG.The recombinant protein was purified using Ni-IDA affinity chromatography and tested using SDS-PAGE electrophoresis. Results In total,2transcripts were obtained.The ORF of Aalb_34ku-2_1was 954 bp,encoding aprotein of318 aa with a pI of 5.84.The ORF of Aalb_34ku-2_2was 882 bp,encoding aprotein of 294 aa with a pI of 6.09.A recombinant Pet28a（＋）-Aalb_34ku-2_1expression vector was constructed and transformed into E.coli BL21（DE3）.The rAalb_34ku-2_1protein was found in inclusion bodies according to SDS-PAGE. Conclusion A recombinant Pet28a（＋）-Aalb_34ku-2_1plasmid expressing a salivary secreted protein of the Guangzhou strain of Ae.albopictus was successfully constructed and a fusion protein was obtained.