Cloning Of Specific Antigen Gene Of Mycobacterium Tuberculosis And Preliminary Study On Nanoemulsion Vaccine

Objective:Tuberculosis is one of the deadly disease that threaten all the world. Despite people were injected with BCG after birth soon, due to immune deficiency and low Immune protection of BCG, the mortality of tuberculosis is increasing which has become an important global public health and social problems. So researching new vaccines of TB has been the emergency work of numerous scholars, in recent years. The key is the choice of vaccine antigens and tuberculosis antigens are mainly CFP10, ESAT-6, Ag85A at the research and development phase. With the development of nanometer technology in biology and medicine research, the improvement of nano adjuvants, and nano drug-loading system, it will brings new breakthrough to the researchers who study TB vaccine. Nanoparticles (NP) has small partical size (1～100nm) , and can distribute specific tissues through human blood capillary, so it can be used for controlled-release, slow-release of drug and the development of single vaccine. So this experiment amplify specific antigen gene, constructing protokaryon recombination plasmid and eukaryotic recombination plasmid.One part, expression protein of protokaryon recombination plasmid was injected to mice intraperitoneal. Another part, with inorganic nanometer (SiNP) packaging eukaryotic recombination plasmid that inject mice muscular and cultivated with cells, to be used to observe the expression of DNA in the cell and muscle.This observation provide experiment basis for the research of nano slow-release vaccine.Method:To amplify ESAT-6, CFP10, MPT51, and PpiA protein cding genes by PCR, The CFP10 gene was selected to construct the original nuclear restructuring plasmid PET-30a-CFP10 and expresspurification protein. To use the Immunized mice with purified protein(40ug/only) (0,21d),while building eukaryotic restructuring plasmid pEGFP-N1-CFP10,preparing nanometer nucleic acid vaccine used to be cultivated With cells 48 hours,another part injected BALB/c mice muscle (50ug/only),Afer 3 days,taking muscle tissue and doing paraffin section, the protein changes was observed in cell and muscle tissue with fluorescence microscope.Result:Successfully obtain nuclear restructuring plasmid PET-30a-CFP10 and purified protein, the fusion of 65nm SiNP with eukaryotic plasmid pEGFP-N1-CFP10 was injected into mice.After, green fluorescence have been observed in the muscle (SiNP parcel red dots and present red fluorescent),the result demonstrate that Nano-DNA was successfully injected into the muscle tissue and red nano luminescent quantum dots were observed.But green fluorescent protein were not expressed in the plasmid. Nano-DNA successfully were induced PK cells to express the GFP.Conclusion:CFP-10 protein was successfully expressed and four kinds of specific antgen gene were cloned.Nano-DNA vaccine SiNP-pEGFP-N1-CFP10 was built and successfully expressed in mouse muscle. The CMS pEGFP-N1 was Constructed and successful expression of GFP in PK cells.