CAJ | 학술논문

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。
근거사안산단백매보수성안기산서렬설계인물,이렴형선두비반포혈자비cDNA위모판,경PCR확증득도SP30기인적보수구,통과5＇RACE（rapid amplification of cDNA Ends）화3＇RACE기술득도전장기인。장해기인련입원핵표체재체pGEX-4T-1,경IPTG유도순화득도중조단백。이렴형선두비란、유비、약비、성비각발육계단적cDNA위모판진행Real-timePCR실험。결과현시,SP30기인전장1194 bp,개방열독광장900 bp,편마299개안기산,예계단백분자질량30 kDa。Real-time PCR분석결과표명,해기인재사개불동적발육계단균유불동정도적표체。
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.