Constructure Of Prokaryotic Expression Vector And Role In Embryonic Implantation And Early Development For Nm23-M2

Objective: By constructing prokaryotic expression vector of nm23-M2, purifying the recombinant protein and fabricating anti-nm23-M2 antigen, its expression, distribution and development change were investigated in embryonic implantation and early development of mice. These results may give us some very important information to better understand the mechanism of embryonic implantation and early development. Method: 1. The genes encoding nm23-M2 was amplified by RT-PCR. The gene was cloned into pQE30 vector (taking unique HindIII and BamHI sites ) by emzyme digestion. The recombinant vector were identified with DNA sequencing and endonucleases. 2. The recombinant proteins were expressed in E.coli.SG13009, identified by SDS-PAGE and western-blot, purified by affinity chromatography. Rabbits were immunizated with the purified protein. Difference between anti-nm23-M2 antigen and anti-nm23-H2 antigen was contrasted. 3. We have set up mouse animal model for pregnant 4d, 6d, 8d and 10d. We have analysed nm23-M2 gene expression of mice endometria in pregnant 4d by RT-PCR and proteins expression of implantation sites and peri-implantation sites in pregnant 6d, 8d, 10d by immunohistochemistry. Result: 1. Recombinant nm23-M2/pQE30 was successfully constructed. The target genes inserted into pQE30 plasmids were confirmed by restriction enzyme analysis and DNA sequencing. 2. Recombinant nm23-M2 protein can be expressed efficiently in E.coli.AG13009. Confirmed by SDS-PAGE and western-blot, the molecular weight of the recombinant protein is 17Kda. The highest expression level of the protein accounted for 45% of total cellular proteins and the purity is more than 97%. By immunized rabbits with the purified proteins, we have obtained the blood serum. The titer of antibody is 1:64. The purity of antibody is over 90% and its concentration is 13.6mg/ml after the blood serum was purified. It is anti-nm23-M2 antibody by western-blot evaluation. Immunohistochemical analysis shows this antibody is more sensitive than anti-nm23-H2 antibody. 3. The results of RT-PCR show that there are nm23-M2 expression in endometria of all pregnant groups and non-pregnant groups. Especially, this expression in pregnant groups is higher than that of non-pregnant groups(P<0.05). Compared between different pregnant groups, there are significantly differences(P<0.01). Besides, our results also demonstrate that nm23-M2 expression at implantation sites is significantly higher than those of the peri-implantation sites( P<0.01). 4. Immunohistochemical analysis showed that there were nm23-M2 expression in endometria of all pregnant groups and non-pregnant groups. The protein expression in pregnant group is higher than that of non-pregnant group(P<0.01). There are significantly differences between different pregnant groups (P<0.01) , and the results showed gradual hoist trend. This result also demonstrates that nm23-M2 proteinexpression at implantation sites is significantly higher than those of the peri-implantation sites( P<0.01). Conclusion: 1. Prokaryotic expression vector of pQE30/nm23-M2 was successfully constructed. 2. Expression and purification of recombinant proteins was successful. This protein can induce specific homoral immune responses in rabbits and produce specific antibody. This result also shows that expression protein has good antigenicity. We have obtained high purity of the anti-nm23-M2 antibody. This antibody has a good immunoreaction, more sensitive than anti-nm23-H2 antibody. 3. nm23-M2 has many biological functions which include control of normal development, differentiation and apoptosis. It may play a different role in different periods of the blastocyst development. The interaction of nm23-M2 with other genes/proteins has an important role during implantation and early development.