Bioinformatics Analysis Of Hepatocellular Carcinoma-Associated Antigen KINECTIN Protein And Prokaryotic Expression Of Gene Fragment KINECTIN

Objective: To ascertain the possibility of hepatocellular carcinomar associated antigen Kinectin used for biological therapies, bioinformatics analysis of kinectin protein were carried out. And bioinformatics analysis results suggest that 2089-2367bp gene fragment of kinectin contains several putative immunogenic epitopes.Then this fragment was amplified and cloned into prokaryotic expressing vector. The expression and preliminary purification of recombinant protein 1-8a were achieved successfully. These provide the basis for the biological therapies of kinectin.Methods: (1)Vector NTI and protein analysis software on Internet were used to analyze kinectin protein sequence and conserved domain;(2)The total RNA of human hepatocellular carcinoma tissue was extracted for synthesizing cDNA by reverse transcriptase. The specific primers were designed according to the cDNA encoding sequence of Kinectin(Z22551)published by GenBank. Then the gene segment of Kinectin(1-8a)was amplified by reverse transcriptase polymerase chain reaction(RT-PCR), and inserted into the expression vector pMAL-C2. The recombinant vector pMAL-C2/kinectin1-8a was transformed into E.coli DH5α. The positive recombinant clones were comfirmed by blue/white secreening assay, PCR assay and DNA sequencing. And the positive recombinant plamid extracted from a positive clone was transfered into the expression host-E.coli Rosetta. 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 supernatant throught the amylose-resin column can be collected and assayed by SDS-PAGE after breaking the induction bacterias by sonication. And the interest protein was separated by factor Xa protease digestion to remove MBP, and identified by protein mass chromatographic analysis.Result:(1)Bioinformatics analysis of Kinectin protein showes that:①Molecular weight of kinectin protein is 156KD, and isoelectric point is 5.52. N-terminal of kinectin protein showes strong hydrophobicity and hydrophobic regions lie in interior of this molecule;②Amino acid sequences of Kinectin among human and 5 different mammals shares high homology. Homology analysis and their homologous levels descend as Vulpes vulpes(93.5%), Bos Taurus(92.6%), Equus caballus(92.5%), Mus musculus(83.3%) and Rattus norvegicus(64.4%);③The secondary structure of kinectin is mainly composed ofα-helix. There are a transmembrane helix(9-29AA), a transmembrane domain and a signal peptide;④Kinectin protein sequences consist of many glycosylation, phosphorylation site. The presence of mulitiple domain in Kinectin protein is located at 2 ~ 699, 776 ~ 899, 923 ~ 1319 amino acid position;⑤There are multiple T cell epitopes and B cell epitopes.(2)The gene segment of kinectin have been successfully inserted into the expression vector pMAL-C2, and the MBP-kinectin 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 4 hours with the final concentration 0.3mmol/L of IPTG. The fusion protein was identified by SELDI-TOF-MS.Conclusion:(1)Bioinformatics analysis of kinectin protein suggested that kinectin protein contains strong immunogenicity and multiple epitopes;(2)Epitope prediction results showed that there are a predicted T cell epitope and five predicted B-cell epitopes with high score in the sequence of 1-8a;(3)Recombinant protein with N-terminal MBP tag encoding by pMAL-C2/kinectin1-8a was successfully expressed and preliminary purified.