Prokaryotic Expression And Identification Of Hepatocellular Carcinoma-Associated Antigen GCF2 Gene Fragment

Objective: To construct a prokaryotic expression vector of pET-30a/GCF2-5a;To expression and identification of recombinant protein 2-5a,provide basis for further study of GCF2.Methods: (1)Software on Internet were used to analyze kinectin protein sequence and conserved domain;(2)The total RNA of human hepatocellular carcinoma tissues was extracted for synthesizing cDNA by reverse transcriptase. The specific primers were designed according to the encoding sequence of GCF2 published by GenBank. Then the DNA segment of GCF2(2-5a)was amplified by reverse transcriptase polymerase chain reaction(RT-PCR). This fragment was inserted in the cloning vector pGEM-T and sequenced. Finally, the cDNA fragment was cloned into pET-30a expression vector. The recombinant vector pET-30a/GCF2-5a was transformed into E.coli DH5α. And the positive recombinant plasmid extracted from a positive clone was transferred into the expression host-E.coli BL21(DE3). The engineering bacteria were induced under different conditions. Then the expression products were analyzed by SDS-PAGE to find out the best induction conditions. At last, the protein was identified by protein mass chromatographic analysis.Results: (1)Bioinformatics analysis of GCF2 protein showes that: Molecular weight of GCF2 protein is 83kDa, and isoelectric point is 4.55. There are no transmembrane region and signal peptide. There are multiple T cell epitopes. (2)The result of electrophoresis showed that GCF2 gene fragment had been cloned into pET-30a vector,and the sequence analysis results showed that sequence of GCF2 gene homolog fragment in recombinant vector was completely identity with GenBank data. The gene segment of GCF2 have been successfully inserted into the expression vector pET-30a, and the His-GCF2-5a protein can be expressed when the engineering bacterias are induced. Determined the optimal induce condition that was firstly shaking 3 hours in 37℃, then in 30℃for 3 hours with the final concentration 1mmol/L of IPTG. The fusion protein was identified by SELDI-TOF-MS.Conclusions:(1)Bioinformatics analysis of GCF2 protein suggested that GCF2 protein contains strong immunogenicity and multiple T cell epitopes;(2)The prokaryotic expression vector of GCF2 gene fragment was successfully constructed. The fusion protein His-GCF2-5a was high efficiently expressed.