[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].

作者信息

Ling Jia-jian, Hou Min, Liu Jian-nan, Zhang Zi-hao, Zhang Yao-juan

机构信息

Department of Pathogenic Biology and Immunology, Nanjing Medical University, Nanjing 210029.

出版信息

Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2003;21(3):144-6.

PMID:14628343

Abstract

OBJECTIVE

To screen and identify the recombinants from the cDNA library of the adult Paragonimus westermani(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 XL1-Blue. The recombinants from positive clones were amplified by PCR, sequenced and cut off by KpmI/BamHI 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 westermani.

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 Paragominus westermani.

CONCLUSION

A recombinant plasmid Pw-2 encodes Pw pre-procathepsin L is constructed.

摘要

目的

筛选并鉴定来自卫氏并殖吸虫成虫cDNA文库的重组体，用于免疫诊断和免疫预防。

方法

用经大肠杆菌XL1-Blue提取物预吸收的卫氏并殖吸虫成虫抗原免疫兔血清（IRS）筛选卫氏并殖吸虫成虫cDNA文库。对阳性克隆的重组体进行PCR扩增、测序，用KpmI/BamHI酶切，然后亚克隆到pRESETB载体中。表达融合蛋白，进行SDS-PAGE分析，并用抗卫氏并殖吸虫虫体抗原的免疫兔血清进行Western印迹鉴定。