长角血蜱保护性抗原基因P_(27/30)的克隆和原核表达  

Cloning and Prokaryotic Expression of P_(27/30) Gene of Parthenogenetic Haemaphysalis longicornis

作　　者：杨彩明[1] 杨光友[1] 张晓谦[1] 贾小勇[1] 余增莹[1] 

机构地区：[1]四川农业大学动物医学院,雅安625014

出　　处：《畜牧兽医学报》2008年第10期1406-1410,共5页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：四川省学术带头人培养基金资助(55035)

摘　　要：采用RT-PCR技术首次从孤雌生殖长角血蜱四川株克隆到P27/30基因,扩增序列全长670 bp,包含完整的开放阅读框,编码201个氨基酸,预测蛋白相对分子质量为23.38 ku。同源性分析表明孤雌生殖长角血蜱中国株与日本株P27/30基因同源性高达99.85%。经RT-PCR检测分析,该基因在孤雌生殖长角血蜱的卵、幼蜱、若蜱、饥饿成蜱和饱血成蜱这几个阶段均有表达。将该基因亚克隆后连接到pET32a(+)原核表达载体,转化BL21(DE3)宿主菌,经IPTG诱导可成功进行表达。表达的目的蛋白大小为24 ku左右,与预期大小一致;Western-blot显示兔抗长角血蜱全虫抗体能够识别该重组表达蛋白。With reverse transcription polymerase chain reaction（RT-PCR）, P27/30 gene was cloned from parthenogenetic Haernaphysalis longicornis of China. The length of P27/30 gene was 670 bp, it included complete ORF and encoded 201 amino acid residues,the deduced mass was 23. 38 kDa. Its nucleotide sequence exhibited 99.85% similarity to that of the P27/30 gene from Japan parthenogenetic Haemaphysalis longicornis. Results of RT-PCR analysis showed that the P27/30 gene could express in the eggs, larvae, nymphs, fame adult ticks and engorgement adult ticks. After subcloned into expressing vector pET32a（＋） ,it could express in E. coli BL21 （DE3） . The recombinant protein was about 24 kDa. With Western-blot analysis, the recombination express protein was approved to be able to recognize antibody of Haernaphysalis longicornis.

关 键 词：孤雌生殖 长角血蜱 P27/30基因 克隆 原核表达 

分 类 号：S852.746[农业科学—基础兽医学] Q786[农业科学—兽医学]