Preliminary Study On Molecular Cloning, Expression And The Function Of HLX In Human

Objective To clone human HLX gene,and to express and purify HLX protein.To prepare the rabbit polyclonal antibody against HLX and confirm the specificity of antibody.To detect the expression of HLX gene on peripheral blood mononuclear cells from gastric carcinoma sufferers by real time quantitative PCR and to analyze the effects of the expression of HLX gene on the balance of Th1/Th2 and the relationship between the expression of HLX gene and gastric carcinoma occurrence.Methods(1) HLX gene was cloned from the mononuclear cells of human cord blood by RT-PCR and constructed into pMD19-T vector.The plasmid was transformed into E.coli DH5αand identified by restriction digestion and DNA sequencing.(2) The full length of human HLX open reading frame gene was cloned from pMD19-T-HLX plasmid by PCR and was constructed into pQE30 vector.The pQE30-HLX plasmid was transformed into E.coli DH5αand identified by restriction digestion and DNA sequencing.(3) The pQE30-HLX plasmid was transformed into E.coli M15.The fusion protein was expressed with isopropylβ-D-1-thiogalactopyranoside(IPTG) and identified by SDS-PAGE and Western blot.The target fusion protein was purified by Profinity IMAC Ni-Charged Resin column.The rabbit was immunized by the target fusion protein.Then the blood serum containing HLX polyclonal antibody was collected.The titer and specificity of antibody was confirmed by enzyme-linked immunosorbent assay(ELISA) and Western blot,respectively.(4) The expression of HLX on peripheral blood mononuclear cells from gastric carcinoma sufferers was detected by real time quantitative PCR and compared with control expression.Moreover,the relationship between T-bet,GATA-3 and HLX were analyzed to understand the disproportion condition on Th1/Th2.Results(1) We cloned HLX gene successfully and constructed the pMD19-T-HLX plasmid.(2) We cloned the HLX open reading frame gene successfully and constructed the pQE30-HLX plasmid.The sequencing consequence demonstrated that the HLX open reading frame gene was 1467bp and encoded 488 amino acids.The target gene shared 100%concordance with the sequence of human HLX in Genbank.We gained the HLX fusion protein that was about 55 kD.(3) We prepared the polyclonal antibody against human HLX,which was approved with high reactivity and specificity by ELISA and Western blot,respectively. And it was approved that HLX protein had high immunogenicity.(4) Real time quantitative PCR results indicated that the expression of HLX on peripheral blood mononuclear cells from gastric carcinoma sufferers decreased compared with control.And we analyzed the correlation between T-bet and HLX and the correlation between GATA-3 and HLX.ConclusionThe pQE30-HLX plasmid was constructed,the fusion protein was obtained from E.coli M15 and the anti-HLX polyclonal antibody was prepared successfully.The expression of HLX on peripheral blood mononuclear cells from gastric carcinoma sufferers decreased compared with control expression by real time quantitative PCR. Moreover,the correlation analysis with other Th1 cytokine suggested that it shares positive correlation with T-bet and negative correlation with GATA-3 in gastric carcinoma.