Cloning And Expression Of The NM23-M1 Gene In E.Coli SG13009 And Study On Its Biological Roles

With the development of cellular biology, molecular oncology, experimental embryology, experimental oncology and immunoembryology, more and more researches have been carried out concerning comparative studies between normal embryonic development and neoplasm progression. Striking similarities between embryonic development and neoplasm progression have been found. Both of them exhibit characteristics of "young" cells in growth and development, and have great potential of proliferation, growth and differentiation. There are similar for genes expression (oncogenes/anti-oncogenes, etco-hormones, ecto-isoenzymes, embryonic antigens), mechanisms of angiogenesis and immunological escaping. Especially, invasion of cancer cell and implantation exist many similarities, both of them possess invasive capacity on ECM. These findings suggest there exists homogeneity between embryo and neoplasm. Studies on homogeneity between embryo and neoplasm were focused on the intersection of oncology and embryology. An appreciation of the mechanisms to control "limited" trophoblast cells invasion may likewise lead to insights of "uncontrolled" invasion and metastasis in malignant cells and lead to efficient approach to control their growth and spread within host tissues. Although nm23 gene is a metastatic suppressor gene, its expression protein NDPK is a multifunctional enzyme which existe cytoplasm and cell membrane. And NDPK have complicated biological function. These suggest that nm23 gene may play an important role during the embryo implantation. Objective: The study aimed to construct the vehicle of prokaryotic expression (pQE30/nm23-M1) and fabricate the polyclonal antibody. We can use the anti-nm23-M1 antibody to investigate the nm23-M1's function during mice's embryo implantation. Methods: 1. Extraction the RNA from mice's brain tissue, and obtained the fragment of nm23-M1 gene by PCR amplification. The nm23-M1 gene's primers designed to generate HindIII and BamHI restriction sites at the 5'and 3'ends of the amplified fragments respectively. The genes were cloned into unique HindIII and BamHI sites of the pQE30. The recombinant plasmids were identified with DNA sequence and endonucleases. 2. The recombinant plasmids (pQE30/nm23-M1) were expressed in E.coli SG13009 and the recombinant protein were purified. Rabbits were immunizated with the purified protein. 3. Blastocyst implantation rate was recorded by injecting different dosage polyclonal antibodies of against nm23-M1 to the early pregnant mice body, and compared the changed regularity of the expressive protein quantity in endometria. Results: 1. Recombinant plasmids (pQE30/nm23-M1) were constructed and the inserted target genes were confirmed by restriction enzyme analysis and DNA sequencing. The fusion gene was sequenced, the mutation rate was 0.4%(2/459)and mutation was nonsense. 2. The recombinant plasmids (pQE30/nm23-M1) were expressed in E.coli SG13009, and its expressive products were analyzed by SDS-PAGEand identified by Western-blot. Antibody titer of anti-nm23-M1 was 1:32. 3. By compared with the control group, the implantation rates in experimental groups(D5) decrease and the expressive protein quantity also decrease in endometria. Conclusion: 1. Recombinant plasmids (pQE30/nm23-M1) were constructed correctly. 2. The dissoluble recombinant protein (pQE30/nm23-M1) can be expressed in E.coli SG13009, and was purified by affinity chromatography. The recombinant protein was used to immunity of rabbits, the purified polyclonal antibodies were obtained. The purity coefficient of polyclonal antibodies is 90%. This result provided condition for investigating the molecular regularity of implantation. 3. By injecting different dosage polyclonal antibodies against nm23-M1 to the early pregnant mice body, we find that implantation rates decrease with increasing antibody quatity. By the analysis of western blot and immunoreactivity, expressive quantity of the protein in endometria decreased with increasing antibody quatity. This investigation suggested that the interactio...