白纹伊蚊(广州株)唾液Aalb_56 ku基因克隆表达与免疫原性分析

CLONING,EXPRESSION AND IMMUNOGENICITY ANALYSIS OF AALB_56 KU GENE OF AEDES ALBOPICTUS( GUANGZHOU STRAIN)

探讨Aalb_56 ku基因在白纹伊蚊(广州株)不同组织中的表达差异;对唾液腺中Aalb_56 ku基因进行克隆表达并探索其免疫原性。采用实时荧光定量RT-PCR法对雌蚊中肠、脂肪体、唾液腺不同组织中Aalb_56 ku基因的表达差异进行分析。针对Aalb_56 ku基因序列设计特异性引物,以唾液腺RNA为模板获得56 ku全长基因序列,构建重组质粒并测序,将测序序列进行生物信息学和抗原表位预测分析。IPTG诱导表达,亲和层析法纯化重组蛋白;制备小鼠特异性抗Aalb_56 ku蛋白抗血清。结果显示,Aalb_56 ku在白纹伊蚊(广州株)唾液腺(SG)中高表达。克隆所获唾液腺56 ku基因全长1 593 bp,可编码530个氨基酸,信息学分析提示该基因含有信号肽序列,等电点为8.40,抗原表位预测提示含有23个表位。p ET30a(+)-56 ku重组质粒可表达约56 k Da大小的融合蛋白,且呈包涵体形式表达,Western blotting鉴定具有His标签。纯化蛋白免疫Balb/c小鼠后可获得1∶100效价的特异性抗血清,提示该重组蛋白具有一定的免疫原性。

To analyze the mRNA level of Aalb_56 ku gene in Ae.albopictus（Guangzhou strain） different tissues.To clone,express the Aalb_56 ku and discuss immunogenicity of the Aalb_56 ku protein.The mRNA level of Aalb_56 ku in midgut（MG）,fat body（FB）,salivary gland（SG） of adult female Ae.albopictus was analyzed by real-time quantitative PCR.The specific primers were designed and Aalb_56 ku gene was cloned from Ae.albopictus （Guangzhou strains） SG by RT-PCR.The recombinant plasmid was constructed and identified by sequencing analysis.The valid plasmids were analyzed by bioinformatics and antigen epitope prediction.The prokaryotic recombinant plasmid pET30a（＋）-56ku was induced to expressed in E.coli BL21（DE3） by IPTG,and the 56ku recombinant protein was purified by Ni-affinity chromatography.The results showed that Aalb_56 ku is expressed significantly higher in SG than that in FB and MG.The length of Aalb_56 ku gene was 1 593 bp,encoding 530 amino acids,containing the signal peptide The isoelectric point of Aalb_56 ku protein was 8.40 and the protein was predicted to 23 epitopes.The pET30a-56ku/BL21（DE3） was successfully expressed and the recombinant 56ku protein was obtained in inclusion form.The anti-56ku serum was obtained from Balb/c mice and the titer was 1：100,suggesting that the protein has a certain immunogenicity.