Respiratory Syncytial Virus M2-1 Gene Expression Vector Construction And M2-1 Protein Polyclonal Antibody Preparation

Respiratory syncytial virus (RSV), which is the leading viral cause of lower respiratory tract disease in infants and children, was well known. In recent years, the infectious adults with RSV have increasingly reported, especially the elderly and the immunocomprimised as well as the patients with chronic cardiopulmonary diseases. So far, It has not effective drugs for the treatment of the patients with RSV infection. Because of the improvement of the patients who were treated with RSV special immulobulin or F,G monoclonal antibodies, or RSV vaccines, It was paid an attention to by investigator. It was reported by Chen HW that the RSV N gene was able to be amplified by RT-PCR in resected carcinomas of the lungs. the finding suggested that there was some relationship between RSV and carcinoma of the lung, but it has been unknown whether it is the potential infection or the cause of lung carcinogenesis. M2- I is a conservative gene. M2- 1 protein is one of neucleocapsid proteins of RSV. Recently, it has been demonstrated that M2- 1 protein has increasing processivity of the viral traitseription, and promotes transcription readingthrough at RSV gene junctions, and play important role to regulate the transcription of RSV mRNA. It were not reported about study of M2-lgene in national medicine, and the relationship between the RSV and the lung cancers in foreign country. The construction of M2-1 gene expressing vector and the preparation of M2-l polycloned antibodies will contribute to explain the functions of M2-1 gene and the relationship between the RSV and the lung cancers, and will provide a new field for study of RSV passive immunity and RSV vaccines. Objectives: In this study, we will, (1) construct M2-1 gene expressing vector and prepare M2-1 protein polycloned antibodies, which will lay foundation for explaining the biolkal functions of M2-1 gene as well as the relationship between the M2-1 gene and the cause of lung carcinogenesis. (2) explore the feasibility of the method of amplifying the M2-1 cDNA by RT- PCR and M2-1 protein as well as M2-1 polycloned antibodies which were applied to clinical detection. (3) open up earlier study for the relationship between the vector and M2-1 protein and vaccine. Methods: (1) The RSV was cultured in Hep-2 cells. After the cells were harvested, the total cell RNA was extracted. The M2- 1 gene cDNK was amplified by RT-PCR. (2) The plasmid pGEX-2T were extracted. The pGEX- 2T and M2-lgene cDNA was digested by EcoRI and XhOI. After digestion, The ligation of pGEX-2T and M2-ii c.DNA was performed. The products were transformed to E.coli BL-2 1. (3) The prokaryotic positive recombinants were screened by PCR and identified by enzymatic digestion. (4) On the basis of construction of M2-l gene prokaryotic expressing vector, The eukaryotic expressing vector was constructed, and identified as the same as prokaryotic expressing vector did. (5) The M2-l protein was induced and purified (6) Preparation of M2-1 polycloned antibodies was performed, and the antibodies were determined by Western blot and ELISA. Results: (1) RT-PCR products were analyzed by agarose gel elecrtrophoresis, The bands were 620bp in size, respectively, and the controls were negative. (2) PCR products were examined by ultraviolet light, and the DNA fragments were 620 bp in size. The controls were negative. (3) Enzyme-digested products were separated by agarose gel elecrtrophoresis,...