Expression,Purification And Immunogenicity Of PE19 Protein From Mycobacterium Tuberculosis

In the genome of Mycobacterium tuberculosis H37Rv,the gene encoding the PE/PPE protein family accounts for about 10%.Previous studies have shown that the PE/PPE proteins are related to a series of immune responses initiated by Mycobacterium tuberculosis infection host.PE19 protein is the compositional protein of the ESX-5 system in the type Ⅶ secretory system of Mycobacterium tuberculosis.The system is related to the secretion of PE/PPE protein of Mycobacterium tuberculosis.In this study,the target gene was cloned into the prokaryotic vector and cloned into the prokaryotic vector.The fusion protein was induced by IPTG.The fusion protein was purified by affinity chromatography and gel filtration chromatography.And then use ELISPOT technology to explore its immunogenicity.First,the 4 recombinant plasmid was constructed by two different cloning methods.The expression of soluble fusion protein was obtained by the small expression experiment.The expression of His6-PE19,which was induced by recombinant plasmid pMCSG7-PE19,was used as antigens to study the immunogenicity of the PE 19.In this study,pMCSG7-PE19 was constructed by Ligation-independent cloning(LIC).LIC has many advantages.For example,LIC can meet the requirements of high-throughput cloning.Plasmids and gene processing are relatively simple.The design of primers does not need to consider the site of the enzyme,just need add a specific sequence at both ends of the upstream and downstream primers.There is no special requirement for insertion of genes.This method not only saves time,but also reduces the cost.Finally,the ELISPOT technique was used to detect whether PE 19 could induce specific cellular immune response,release interferon-y,and explore the immunogenicity of PE 19 protein.In this study,the detection rate of PE 19 was equal to the detection rate of antigen B,suggesting that PE 19 has the potential to be a specific immunoreactive agent in ELISPOT.A small number of inconsistent samples were found in the patient samples,indicating that PE 19 had the potential to be a supplement or supplemental peptide for antigen A and antigen B.Due to the limited number of samples,the sensitivity and specificity of the immunogenicity of PE 19,the need to expand the number of experimental samples in order to get more stringent conclusions.Experiments show that PE 19 can indeed be used as antigen,causing tuberculosis infection of the body to produce a specific immune respons.