【摘要】 通过对饥饿的长角血蜱雌性成蜱cDNA表达文库中筛选到的未知基因进行5′RACE扩增,以期获得长角血蜱MLP基因全长cDNA序列。设计通用引物,采用PCR方法分别从青海血蜱、麻点璃眼蜱、森林革蜱、微小牛蜱及小亚璃眼蜱中对MLP基因完整的开放阅读框进行了扩增,并将其克隆到表达载体pGEX-4T-1中,构建重组质粒,转入BL21(DE3)中,经IPTG诱导表达,分别用SDS-PAGE和Western-blot分析目的蛋白的表达情况和反应原性。结果表明,成功扩增了长角血蜱的MLP基因全长序列及多个蜱种的MLP基因开放阅读框序列,体外高效诱导表达了约39.2ku的不同蜱的MLP重组蛋白。Western-blot结果表明,长角血蜱的MLP重组蛋白具有很强的反应原性,且不同蜱的重组蛋白间具有很强的交叉反应性。更多还原

【Abstract】 The full-length cDNA encoding muscle LIM proteins(MLP) from Haemaphysalis longicornis,which was screened from a cDNA library constructed from unfed female ticks,was obtained by 5′rapid amplification of the cDNA ends(5′RACE).The nucleotide sequence of MLP gene and the deduced amino acid were analyzed,and the complete open reading frames(ORF) were amplified by PCR using universal primer from ticks H.qinghaiensis,Hyalomma anatolicum anatolicum,H.rufipes,Dermacentor silvarum and Boophilus(Rhipicephalus) microplus,respectively.The PCR product was cloned into expression vector pGEX-4T-1 to construct recombinant plasmid which was then transformed into Escherichia coli BL21(DE3) for expression.The expression and reactionogenicity of recombinant protein were analyzed by SDS-PAGE and Western-blot,respectively.It was found that the expressed fusion protein was approximately 39.2 ku in molecular weight.Western-blot analysis showed that rabbit antiserum against the adults of H.longicornis could recognize the rHLMLP and the protein of different ticks.The results suggested that rHLMLP could be used as a potential candidate universal antigen for the development of anti-tick vaccines.更多还原