镰形扇头蜱丝氨酸蛋白酶SP30基因的克隆及原核表达

CLONING AND PROKARYOTIC EXPRESSION OF SERINE PROTEASE SP30 GENE OF RHIPICEPHALUS HAEMAPHYSALOIDES

根据丝氨酸蛋白酶保守性氨基酸序列设计引物,以镰形扇头蜱半饱血雌蜱cDNA为模板,经PCR扩增得到SP30基因的保守区,通过5'RACE(rapid amplification of cDNA Ends)和3'RACE技术得到全长基因。将该基因连入原核表达载体pGEX-4T-1,经IPTG诱导纯化得到重组蛋白。以镰形扇头蜱卵、幼蜱、若蜱、成蜱各发育阶段的cDNA为模板进行Real-timePCR实验。结果显示,SP30基因全长1194 bp,开放阅读框长900 bp,编码299个氨基酸,预计蛋白分子质量30 kDa。Real-time PCR分析结果表明,该基因在四个不同的发育阶段均有不同程度的表达。

The specific primers were designed using Genetyx software according to the conserved nucleotide sequence of serine protease of Rhipicephalus haemaphysaloides.The conserved region of SP30 gene was amplified by PCR using cDNA template from feeding female R.haemaphysaloides.The full length of SP30 gene was obtained by 5′rapid amplification of cDNA ends（RACE） and 3′RACE protocol.The SP30 gene was then sub-cloned into vector pGEX-4T-1 and the recombinant protein was expressed in E.coli BL21 with the induction of IPTG.The resulting SP30 gene contained 1194 bp and the open reading frame contained 900 bp encoding 299 amino acid residues（predicted molecular weight 30 kDa）.Real-time PCR was performed using cDNA templates from eggs,larvae,nymphaea,adults.The results indicated that SP30 gene was expressed differently in four development stages.