白纹伊蚊Rh类糖蛋白基因的克隆与生物信息学分析

Cloning and Bioinformatics Analysis of A Rhesus-like Glycoprotein from Aedes albopictus

目的应用简并引物RT-PCR和RACE方法获取白纹伊蚊Rh类糖蛋白(Aedes albopictus rhesus-like glycoprotein,AaRh)基因的全长cDNA。方法根据冈比亚按蚊、埃及伊蚊等亲缘关系较近物种的Rh类糖蛋白同源性分析结果,在氨基酸高度保守区域184/343和219/337氨基酸位点设计2对简并引物,以白纹伊蚊雌蚊总RNA为模板,应用巢式RT-PCR扩增AaRh的基因片段。根据获得的AaRh基因部分序列,设计2对特异性引物AaRh GSP1、GSP2和AaRh GSP3、GSP4,应用5’RACE和3’RACE分别扩增AaRh基因的5’端和3’端cDNA片段,然后拼接出全长cDNA序列。通过在线生物信息学分析(NCBI和Expasy),对目的基因序列进行生物信息学分析。结果应用2对简并引物进行巢式RT-PCR,获得379 bp AaRh基因片段。应用5’RACE和3’RACE方法,分别获得AaRh基因5’端1008 bp、3’端822 bp cDNA序列,根据两个片段的首/尾共同序列拼接为1717 bp的基因片段。该核苷酸序列经BLASTn分析显示,与埃及伊蚊Rh蛋白的一致性高达95%,鉴定其为白纹伊蚊Rh类糖蛋白基因。AaRh基因具有完整的开放阅读框,其ORF从第128位到1516位含1389bp,编码462个氨基酸。Expasy在线生物信息学程序分析显示,白纹伊蚊Rh类糖蛋白是一个整合膜蛋白,跨膜11次,58aa-446aa具有铵离子通道的结构功能域;理论等电点(pI)5.37,分子量49775.10 Mr,1aa-26aa可能为分泌信号肽序列;含有4个潜在的天冬酰胺糖基化位点和17个线性抗原决定簇,翻译后可能进行糖基化修饰,提示其为糖蛋白。结论成功获取AaRh基因的全长cDNA,生物信息学分析结果为AaRh蛋白的生物学特性和功能研究奠定了基础。

Objective To obtain a full length cDNA sequence of the Rh-like glycoprotein gene from Aedes olbopictus （AaRh） by degenerate RT-PCR and Rapid Amplification of cDNA Ends （RACE）. Methods Based on the highly conserved homologous amino acid sequences of Rh-like glycoproteins from Anopheles gambiae and Aedes aegypti, we designed 2 pairs of degenerate primers on 184/343 aa and 219/337aa. Using total RNA from the adult female of Ae.albopictus as templates, we performed nest RT-PCR to obtain a fragment of the AaRh gene. According to the obtained partial sequence of the AaRh gene, we designed 2 pairs of gene-specific primers （AaRh GSP1 ,GSP2 and A aRh GSP3, GSP4）, and performed 5 ＇RACE and 3＇RACE to amplify 5 ＇cDNA end and 3＇cDNA end of the A aRb gene. Then we spliced a full length cDNA sequence of the AaRh gene. We used BLAST program from NCBI website and programs from Expasy website to analyse the obtained gene sequence. Results Using 2 pairs of degenerate primers to perform nest RT-PCR, we obtained a gene fragment of 379 bp. By 5＇RACE and 3＇RACE, we obtained two gene fragments of 1008 bp and 822 bp respectively. According to the sequences of these two gene, we spliced a gene sequence of 1717 bp. By BLASTn search on NCBI website, it had 95% identity with Rh-like glycoprotein from Aedes aegypti. So it was identified as a full length cDNA sequence of the AaRh gene. The 1717 bp cDNA comprised an ORF from 128 to 1516 encoding a putative protein of 462 amino acids. Bioinformatics analysis by programs on Expasy website showed that A aRh glycoprotein might be an integral membrane protein with 11 transmembrane helixs. The predicted AaRh amino acid sequence was found to have a conserved domain of an ammonium transporter at 58- 446aa, with a predicted molecular weight of 49775.10 Mr and a theoretical pI of 5.37. The possible signal sequence cleavage site of AaRh was after Phe26. There were 17 potential epitopes and 4 potential N-glycosylation sites, suggesting that the AaRh protein should be a glycoprotein. Conclusion We have obtained a full length cDNA sequence of the Rh-like glycoprotein gene from Ae.albopictus. Bioinformatics analysis would help biological characterisation and functional study on this protein.