长角血蜱4D8基因的克隆与原核表达

根据GenBank上登录的蜱4D8基因序列设计引物,用PCR技术从长角血蜱雌蜱研磨组织cDNA中扩增出4D8基因,将其连入pGEM-T Easy载体,构建重组克隆载体pGEM-T-4D8,并对检测为阳性的克隆进行测序。将该基因重组至原核表达载体pGEX-4T-1,经表达纯化后,进行Western-blot试验鉴定其生物学活性。结果,长角血蜱4D8基因的氨基酸序列与青海血蜱、肩突硬蜱的同源性分别为90%、81%。表达的重组蛋白是分子质量约为41ku的融合蛋白。Western-blot试验证明,该重组蛋白能很好地识别兔抗长角血蜱全蜱阳性血清。结果表明,4D8基因作为一种重要的分子标记基因在蜱的流行病学调查方面有着重要的价值,同时作为潜在的候选抗原基因在疫苗的研究方面也有重要意义。

Cloning and prokaryotic expression of 4D8 gene of Haemaphysalis longicornis

To reveal the protective antigen 4D8 immunogenicity of Haemaphysalis longicornis,one pair of specific primers were designed according to the sequence of 4D8 gene of the pathogenic parasites available in GenBank.The 4D8 gene was amplified by PCR from cDNA grinding tissue of female H.longicornis and cloned into pGEM-T Easy vector to construct recombinant clonal vector pGEM-T-4D8,sequenced and analyzed.A recombinant expression plasmid pGEX-4T-1-4D8 was constructed by subcloning the cloned 4D8 gene into the linearized pGEX-4T-1 vector.Then the plasmid pGEX-4T-1-4D8 was expressed in Escherichia coli(BL21).The fusion protein was purified and checked by Western-blot.Results showed that the nucleotide sequence of the 4D8 gene shared 90% and 81% identity with that of H.qinghaiensis and Ixodes Scapularis,respectively.SDS-PAGE analysis showed that the expressed fusion protein was 41 ku in molecular mass.The protein could be recognized by the positive serum in the Western-blot test.In conclusion,4D8 gene was of important significance as a good antigen gene for epidemiological investigation of H.longicornis and development of candidate vaccines against H.longicornis.