[Cloning, ligation and expression of the variable region genes of the monoclonal antibody against human HnRNPA2/B1].Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2004 Dec; 21(6):548-51.ZY
To clone the variable region genes of the monoclonal antibody (McAb) against human heterogeneous nuclear ribonucleoprotein A2/B1 (HnRNPA2/B1), ligate them to assemble single chain Fv (ScFv) gene and express in Escherichia coli.
The specificity of the anti-HnRNPA2/B1 McAb 3E8 to synthetic HnRNPA2/B1 peptide, HnRNPA2/B1 protein in lung cancer cells were examined by dot-immunobinding assay, Western blot and immunohistochemistry. The variable region genes of heavy chain (VH) and light chain (VL) were amplified from hybridoma cell by reverse transcription-polymerase chain reaction(RT-PCR), and then were linked by a linker peptide using SOE-PCR (splicing by overlap extension-PCR) to construct recombination ScFv gene. The latter was cloned into the expression vector pET28 (a+) and expressed in E coli BL21. The expressed product was identified by SDS-PAGE and competitive ELISA inhibition test.
It was shown that the McAb combined specifically with synthetic HnRNPA2/B1 peptide and HnRNPA2/B1 protein in three lung cancer cells. The cloned VH gene and VL gene were 345 bp and 309 bp respectively and were linked successfully to obtain ScFv gene. The ScFv protein was expressed in the form of inclusion body, with molecular weight of 28,000 and immunoreactivity to HnRNPA2/B1.
VH gene, VL gene and ScFv gene of anti-HnRNPA2/B1 antibody were cloned, constructed and functionally expressed in E coli. These results provide the experimental basis for elucidating the role of HnRNPA2/B1 in lung cancer.