[Cloning, ligation and expression of the variable region genes of the monoclonal antibody against human HnRNPA2/B1].
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Wang
Xia
X
Department of Immunology, School of Preclinical and Forensic Medicine, Sichuan University, Chengdu, Sichuan, 610041 P. R. China.
Peng
Xiao-dong
XD
Li
Guang
G
Hu
Li-juan
LJ
Bi
Jian-hong
JH
D000230:Adenocarcinoma / Q000378:metabolism / Q000473:pathology; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D018288:Carcinoma, Small Cell / Q000378:metabolism / Q000473:pathology; D002294:Carcinoma, Squamous Cell / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B / Q000276:immunology*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics; D008175:Lung Neoplasms / Q000378:metabolism* / Q000473:pathology
D000911:Antibodies, Monoclonal; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; C055966:immunoglobulin Fv