Gene Cloning, Expression And Preparation Of Polyclonal Antibody Of The Extracellular Fragment Of HLA-A～*0201

Objective To express and identify recombinant extracellular fragment of HLA-A~*0201 protein in E.coli,and prepare the anti- HLA-A~*0201 polyclonal antibody,this can provide basic for deep study the association between HLA-A~*0201 gene and the biotherapy of lung cancer.Methods The objective gene was amplified by PCR from recombinant plasmid pcDNA3.1—HLA-A~*0201,and cloned into prokaryotic non-fusion expression vector pBv220.Then the recombinant plasmid was transformed into E.coli.After screened by amplicillin, the positive clone was selected. Then the restriction enzymes and sequencing were used to decide it. The positive clone was induced to express in E.coli. The expression product was detected by SDS-PAGE and identified with Western blot analysis. To obtain purified protein, ion exchange chromatography and gel filtration were performed.The purified protein was used as a immunogen to mice,the efficiency of the antibody was determined by the method of ELISA. Results The extracellular fragment of HLA-A~*0201 gene was cloned successfully, the sequencing result was consistent with the foregone sequence exactly. The objective gene was expressed in the prokaryotic system in the form of inclusion...