卫氏并殖吸虫基因片段的克隆与鉴定

Cloning and Identification of the Gene Fragments of Paragonimus westermani

目的 从卫氏并殖吸虫成虫cDDA文库中筛选并鉴定可用于免疫诊断和免疫预防的基因克隆。方法采用预吸收成虫抗原免疫兔血清(IRS,多抗)筛选阳性克隆,经PCR扩增后测定其插入片段的大小。序列分析后,对筛选的重组子进行双酶切,亚克隆入表达载体pRESETB,并转化大肠杆菌BL-21细胞,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,对表达产物进行SDS-PAGE和Western blotting分析鉴定。结果 所筛选的阳性克隆插入片段约800bp。DNA序列分析显示其为编码半胱氨酸蛋白酶族组织蛋白酶L的基因序列。Pw-2重组子特异性表达产物约为32kDa,可被卫氏并殖吸虫免疫兔血清特异地识别。结论 从卫氏并殖吸虫成虫cDNA文库筛选的重组质粒Pw-2编码属于半胱氨酸蛋白酶族组织蛋白酶L。

Objective To screen and identify the recombinants from the cDNA library of the adult Paragonimus west-ermani (PwA) for immunodiagnosis and immunoprophylaxis. Methods PwA cDNA library was screened with the PwA antigen immunized rabbit sera(IRS) pre-absorbed by the extract of E. coli XLl-Blue. The recombinants from positive clones were amplified by PCR, sequenced and cut off by KpnI/BarnHI and, then sub-cloned into pRESETB vector. The fusion protein was expressed,analysed by SDS-PAGE and identified by Western blotting with immune rabbit serum against worm antigen of Paragonimus westerrnani. Results The inserted cDNA fragment from the positive clone Pw-2 was about 800 bp, which contained an open reading frame(ORF) encoding Pw pre-procathepsin L belonging to cysteinase family. Expression product of Pw-2 was a fusion protein of 32 kDa, which can be recognized by immune rabbit serum against worm antigen of Paragonimus 晈esterrnani. Conclusion A recombinant plasmid Pw-2 encodes Pw pre-procathepsin L is constructed.