Chinese Journal of Lung Cancer (Jun 2008)
Construction and expression of recombinant prokaryotic vector PGEX-4T-1-BC006151 correlated with multidrug resistant of lung adenocarcinoma
Abstract
Background and objective The drug resistance to chemotherapeutics is one of the important causes of low survival rate of lung cancer patients. Our previous study has demonstrated that BC006151 is a gene correlated with multidrug resistance of adenocarcinoma of lung. The aim of this study is to clone the BC006151 gene, and to construct recombinant prokaryotic vector PGEX-4T-1-BC006151, and to express it in E. coli BL21. Methods The primer was designed with restriction endonuclease position, then amplified BC006151 by RT-PCR, cleaved BC006151 cDNA and PGEX-4T-1 by BamH and EcoR I. linked it with PGEX-4T-1. Then the two fragments were linked by T4DNA. The post-linked vector was transformed into E. coli. DH5 and then expressed. Transformed the recombinant plasmids containing the correct clone into E. coli BL21 and protein was highly effective expressed. The production of GST fusion protein was identifisd by SDS-PAGE and Western-Blotting. Results The sequence of BC006151 was amplified and identified with that published in GenBank. The prokaryotic expression plasmid PGEX-4T-1-BC006151 was constructed successfully. And a new fusion protein with relative molecular mass of 13 KD was highly effectively expressed in E. coli. Conclusion The BC006151 gene correlated with multidrug resistance of lung adenocarcinoma is successfully cloned and expressed, which is helpful for the preparation of monoclonal and polyclonal antibodies.
