阴道毛滴虫黏附蛋白ap33基因克隆及其表达

CLONING AND EXPRESSION OF THE AP33 GENE OF TRICHOMONAS VAGINALIS

通过提取阴道毛滴虫mRNA ,逆转录合成cDNA ,PCR得到预期长度片段后 ,将其克隆到pUCm-T载体中进行PCR、酶切及测序分析 ,并与GeneBank中核苷酸序列进行同源性分析。再将其克隆至表达载体PET-32a。重组子用酶切、PCR和测序鉴定后 ,转化大肠杆菌并以异丙基 -B- D- 硫代半乳糖苷 (IPTG)诱导表达。从阴道毛滴虫临床分离株中扩增出 92 7bp的ap33的基因片段 ,构建重组质粒PET- 32a (+) - ap33;IPTG诱导后 ,SDS -PAGE显示表达产物的大小约 5 0kDa。

The ap33 gene was amplified from the cDNA of Trichomonas vaginalis by PCR and recombined with plasmid pUCm-T.The recombinant plasmid pUCm-T was identified with PCR,restriction endonuclease analysis and sequencing.Then the ap33 gene was recombined with plasimd PET-32a(+) and was identified with PCR,restriction endonuclease analysis and sequencing.The gene structure was analyzed by comparing with sequences available in the GenBank.The EcoRⅠ/XhoⅠ restricted fragments confirmed by PCR and EcoRⅠ/XhoⅠ digestion,were cloned into expression vector PET-32(a)(+) and the recombinants were transformed into E.coli BL21. Fusion expression was induced by isopropy-beta-D-thiogalactoside(IPTG).Results show ap33 gene was cloned correctly into vector PET-32a(+).The cDNA sequence homology was 99%、97%、94% respectively compared with the gene of Trichomonas vaginalis adhesin protein ap33-1、Trichomonas vaginalis adhesin protein ap33-3、Trichomonas vaginalis adhesin protein ap33-2.High expression was obtained in PET-32 a(+)/ap33/E.coli BL21.