Cloning And Expressing Of HLA-G1 Fusion Gene In E.coli And Studying Of Recombined HLA-G1 On Purification And Function

HLA-G is a non-classical MHC classical I molecule involved in immunotolerance. The induction of immuno-tolerace of foreign grafts is the key to long-term survival of transplanted tissue. The hyperacute rejection of foreign grafts has been greatly inhibited in xenotransplantation by eliminating the natural antibody and by using inhibitor complements. But due to the presence of delayed xenogranfts rejection, the problem of immunological rejection in transplantation has not been completely dissolved.Pregnancy in human and mammanls presents intriguing evidence of natural immuno-tolerance of semiallogentic grafts. HLA-G, which is high expressed on the cyto trophoblast cells at the maternal-fetal interface, plays an important role in the maternal-fetal tolerance and in protecting the fetal not being rejected by maternal by inhibiting the cytolysis of NK cells in the deciduas of uterus. However there is not a clear conclusion that which special conformations or amino acids interact with the special receptors on NK cells. The purpose of our research is to find out the mechanism that interacting of them by mutating some amino acide(s) in HLA-G1 molecule and then observing the changes in affinity and activity between them.It was reported that CTL cells could be easily signed and recognized during MHCs combined with TCR when MHCs converged to a tetramer which was made as below. When gene of BirA enzyme substrate pepties (BSP) was connected with gene of MHC in the tail of carboxy with genetic engeering technics the BSP would conncet with biotin firmly. Because of it multivalue character the biotin molecule could connect four BSP molecule. However reports are not observed whether it is suit for HLA-G and whether HLA-G remains the function after treated with the technic. The purpose of our study is to confirm the infer and do some foundation work about the mechanism between HLA-G and its receptors.In the experiments genes located in extramembrance region of HLA-G1 (simple as H)and BSP(simple as B) were cloned with PCR respectively, and they were connected to a combined gene (simple as HB)by technic of SOE PCR. Then the combined gene was cloned into expressing vector pET28a. After identify with restrict endonucleases cleavage analysis, PCR annlysis and sequence confirmed it was proved that combined expressing vector pET28a-HB is constructed successfully.In the optimized expressing condition both strains, DE3-pET28a-HB and DE3-pET23a-h β 2M,were incurated at the presence of 1mmol/L Isopropyl-β-D- Thiogalactoside (IPTG) at 37°C for 5 and 3 hours respectively. Both of the proteins could be greatly expressed in inclusion body and the two proteins molecule weight was about 36kD and 11kD. HB could react with monoclone antigen 87G while h β 2M with multiclone antigen which is made from ascites in Western blot analysis. Inclusion body contained HB protein could be well pured with Co2+ resin after lysised in 6mol/L urea. And inclusion body contained hβ 2M could be used to help HB refolding after it be washed several times and be treated with 6mol/L urea. The content of protein h β 2M reached about 30% in all proteins in the inclusion body.Protein h P 2M was refolded with dialysis while protein HB with dilution at the presence of h P 2M and nonapeptide. After being condensed with ultrafiltration protein HB was added to the mixture cells of NK92 and K562 at a final concentration 10 μ g/ml in the solution. The lysis rate of NK92 to K562 was measured with LDH method at the rate of 5:1 and 2.5:1 respectively for NK92:K562. The results showed that protein HB inhibited the lysis of NK92 to K562 cellsapparently at a final concentration of 10 μ g/ml.