CDNA Clone And Procaryotic Expression Of Human NIT1 Gene And Preparation Of Rabbit Polyclonal Antibody Against Human NIT1 Fusion Protein

Background and Objective: At present, lung cancer has become the most common malignant tumor threatening human health and life in the world. The mortality and mobidity has been increasing fast during the past 50 years. It becomes the first killer of cancer-related death in many big city in China. Therefore, studying and understanding the molecular mechanism of carcinogenesis in lung cancer are becoming the very important work. The essential characteristics of malignant tumor cell are the unrestrictedperliferation and the suppression of apoptosis. It has been proved that the abnormal activation of oncogene and the inactivation of tumor suppression gene are the important molecular mechanism of oncogenesis and development of lung cancer. It has demonstrated that FHIT gene is a very important tumor suppression gene because it can suppress the growth of tumor cell and promote apoptosis of tumor cell. The abnormality of FHIT gene is probably the early molecular event of carcinogenesis of lung cancer. Therefore, studying and demonstrating the relationship of FHIT gene with carcinogenesis and progression of lung cancer has an important clnical significance for lung cancer. It can reveal the regulative mechanism of cell singnal transduction about FHIT gene in lung cancer. On the other hand, it also can provide the base of theory and experiment for exploring the target drug of killing tumor cell and gene therapy of lung cancer. Our research group leaded by Dr. Zhou QingHua, keep on studying the structures and expression from DNA, RNA to protein level of FHIT gene in the area for a long time, demonstrated that the FHIT gene plaied an important role in carcinogenesis and progression in lung cancer. Transfecting wild type FHIT gene into lung cancer cell A549 cell line can reverse the malignant and metastatic phenotype of lung cancer by in vivo and ex vivo experiments. On the base of our previous research results, the relationship between FHIT gene and NIT1 gene , the candidate downstream gene of FHIT, will be explored. The objective of this study are as follows: â‘  To clon and construct the procaryotic expression vectors of human NIT1 gene by RT-PCR and directive clon techniques; â‘¡To get stable expressive fusion protein of NIT1 in the Rosetta^TM (DE3) ; â‘¢To obtain high quanlity of polyclonal antibody of NIT1 from rabbit immunized with NIT1 fusion protein;d)To determine the location of NITl protein in human placenta tissues by immunohistochemistry.Materials and methods:In this study, two primers were designed according to the human NITl coding sequence from GenBank. EcoR I and Not I sites of restriction endonucleases were introduced to the 5' and 3' terminal primers respectively. Total RNA was isolated from placenta tissues. The fragments of NITl gene encoding NITl protein was amplified by RT-PCR and was inserted into pET-32a vector, which will produce fusion protein of NITl, then the sequence of the recombinant was determined by DNA sequencing. The competent cells of E. coli JM109 were transformed with pET-32a/NITl. The transformation was digested and identified with EcoR I and Not I . Kcoli strain Rosetta? ( DE3 ) were transformed with pET-32a/NITl. IPTG was added to induce the fusion protein expression. The molecular weight of the fusion protein was detected by SDS-PAGE identification. The NITl protein was expressed as inclusion bodies proved by solubility analysis of fusion protein. 6XHis-tagged NITl fusion protein was purified by metal chelate affinity chromatography by the HisTrap?Kit. The NITl fusion protein was analyzed by SDS-PAGE. The rabbits were immunized in the test group with mixture of Nitl fusion protein and complete Freund' s adjuvant (the ratio was 1:1 ) every 3 weeks for 4 times by subcutaneous injection. PBS solutions was injected in the control group every 3 weeks for 4 times by subcutaneous injection. Blood was collected from the rabbit at 10 days after the fourth immunication. The titer and specificity of NITl antibody was determined by indirect ELISA -. Western blot % immunohistochemistry analysis.The results of the study firstly showed as follows in the world:1. NITl gene cDNA was successfully cloned from total RNA of human placenta tissues. The NITl cDNA was recombined into the pET-32a vector . The recombinant was determined by DNA sequencing technique.2. The recombinant pET-32a/NITl was transfered into Rosetta? (DE3), and IPTG was added to induce expression of NITl fusion protein. The NITl fusion protein was identified by SDS-PAGE method. The objetive protein expressed with the manner of inclusion bodies was determined and purified by HisTrap?Kit metal chelate affinity chromatography . A high quantity fusionprotein was obtained.3. The rabbits were immunized with purified Nitl fusion protein. Sera of rabbits was collected. The titer of fusion protein of NITl was from 1:200 to 1:3200 dilution by indirect ELISA . Positive expression can be observed by Western blot when the sera was diluted at 1:200 ratio. Rabbit polyclonal antibody was successfully prepared.4. NITl protein was observed to be located in cytoplasm of villus cell of placenta tissues by immunohistochemical analysis, which is correspondence with location of FHIT protein in cell.Conclusions: (1) Procaryotic recombinant expressive vector of NITl gene was successfully constructed by RT-PCR and directive clonal techniques. (2) Fusion NITl protein was stable expressed in the Rosetta? (DE3). (3 ) High quanlity of polyclonal antibody again3t NITl was obtained from the rabbit immunized with NITl fusion protein. (4) The NITl protein was located in the cytoplasm of villus cell of human placenta tissues.