卫氏并殖吸虫基因片段的亚克隆及其表达的研究

Studies on the Subcloning and Its Expression of the Genes Fragment of Paragonimus westermani

目的:获得诊断肺吸虫病的特异性重组抗原,以了解其作为免疫诊断抗原的价值。方法:将免疫筛选获得的卫氏并殖吸虫基因克隆双酶切,所得cDNA片段亚克隆入表达载体pRESETB,经异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达,以聚丙烯凝胶电泳(SDS-PAGE)和免疫印迹法(Westernblotting)对表达产物进行鉴定。结果:成功地将文库中2个大小不同的卫氏并殖吸虫基因片段连接到载体中。其中Pw-2重组子特异性表达产物是分子质量约为32ku的蛋白带,可被卫氏并殖吸虫免疫兔血清特异性识别。结论:成功构建了编码卫氏并殖吸虫特异性抗原的重组克隆Pw-2。

Objective:To obtain the recombinant antigen of Paragonimus westermani(Pw) for the immunodiagnosis of paragonimiasis.Methods:The cDNA library about Pw was screened and the positive clones from immunoscreening were cut with KpnI and BamHⅠ. Then, the cDNA fragments from cutting were subcloned into vector pRESETB and the expression of subclone products induced by IPTG were analysed by SDS-PAGE and Western blotting with immune rabbit sera against Pw adult worm antigen.Results:Two different fragments of Pw cDNA genes were successfully subcloned into pRESETB. The specific expressive product of recombinant clone Pw-2 was a kind of protein with a molecular weight of 32ku.The fusion protein can be recognized by immune rabbit serum against adult worm antigen of Pw. Conclusion:The recombinant plasmid Pw 2 encoding Pw specific antigen was obtained successfully prepared.