Construction Of A Eukaryotic Expression Vector Of Mycobacterium Tuberculosis 38 KD And ESAT-6 Protein And Its Secretory Expression In HEK 293T Cells

Mycobacterium tuberculosis(MTB)is an obligate pathogenic bacterial species in the family Mycobacteriaceae and the causative agent of tuberculosis(TB).TB is still an important infectious disease.According to the WHO,there are about 9 million new cases occur each year,at least 3 million people die of TB.Tuberculosis is one of China's major public health problems.According to the 2015 WHO estimates,China has the world's third largest number of tuberculosis cases,behind only India and Indonesia,comprising around 10%of the world total.Fast and exact diagnosis of tuberculosis(TB)is one of the important measures to global TB control.38KD protein of mycobacterium tuberculosis is of value for immunodiagnosis of tuberculosis,because of its higher specificity and sensitivity in a variety of tuberculosis related antigen.And the early secretory antigen target(ESAT-6)of mycobacterium tuberculosishas causes strong cellular immunocompetent,especially T cell immunity,so it can maintain constant immune memory for a long time.Thus,both the 38KD protein and the ESAT-6 can be expected to become specific tuberculosis diagnostic reagents and vaccines development.This study is divided into three parts.First of all,we constructed a robust expressing vector of mycobacterium tuberculosis 38 KD proteins,which is able to induce high efficient and secretory expression of 38 KD proteins in HEK-293T cells.The 38KD gene of mycobacterium tuberculosis is synthesized and cloned into T-vector,and then subcoloned into the pcDNA3.0 vector.The recombinant pcDNA3.0-38KD was verified by enzyme digestion and then transfected into HEK-293T cells by polyethylenemine(PEI)-based transfection.24 hours after transfection,the media were changed to serum-free DMEM media for another 3 days.Then the transfected cells and supernatants were collected separately for the detection of 38 KD proteins by Western Blot.Results showed that we have successfully obtained a robust mycobacterium tuberculosis 38 KD protein expressing vector,pcDNA3.0-38KD,which can elicit high level expression of 38 KD protein in both cell supernatants and lysates in HEK-293T cells.Thus,high level of secretory mycobacterium tuberculosis 38KD protein was successfully expressed in mammalian cells.Next we constructed a 38 KD protein and eGFP co-expressing lenti-viral vector,and investigate whether IRES-eGFP sequence have any effect on 38KD protein expression.pLV-38KD-IRES-eGFP was double digestion with Mlu I and Sal I enzyme,removing the segments of IRES-eGFP.The recombinant plasmid was verified by Spe I digestion and then transfected into HEK-293T cells by polyethylenemine(PEI)-based transfection.24 hours after transfection,the media were changed to serum-free DMEM media for another 3 days.Then the transfected cells and supernatants were collected separately for the detection of 38 KD proteins by Western Blot.We have successfully obtained a robust mycobacterium tuberculosis 38 KD protein lenti-viral expressing vector,which can elicit the expression of high level of 38 KD proteins in cell in transfected HEK-293T cells.High level of secretory mycobacterium tuberculosis 38KD protein was successfully expressed in mammalian cells.And the results also showed that IRES-eGFP have inhibitory effect on 38KD protein expression.Based on the results of the second part,we continue to explore the effect of IRES-eGFP segment on the expression of other genes.In a similar way,we constructed a mycobacterium tuberculosis ESAT-6 gene and eGFP co-expressing lenti-viral vector.Results showed that high level of secretory ESAT-6 protein was successfully expressed in HEK-293T cells.And the results also showed that IRES-eGFP have inhibitory effect on 38KD protein expression.Further more,we validated the immunogenicity and immunoreactivity of 38KD protein and ESAT-6 protein through ELISA.The pLV-38KD and pLV-ESAT-6 plasmids were transfected into 293T cells,pLV-eGFP plasmids was used as negative control.24 hours after transfection,the media were changed to serum-free DMEM media for another 3 days.Then the supernatants were collected for ELISA detection.7 TB positive serum samples were detected,results showed that the positive rate for 38KD protein was 71.43%while ESAT-6 protein was 100%,which suggested that both ESAT-6 protein and 38KD protein were effective in TB serological diagnosis.In conclusion,we successfully constructed mycobacterium tuberculosis 38 KD protein and ESAT-6 expressing vector,respectively.Through transfection into HEK-293T cells,both vector can efficiently produce secretory and robust protein in mammalian cells,to our surprise,the expression of both protein were inhibited by the IRES-eGFP segment,while the reason remains to be studied.The immunogenicity and immunoreactivity of 38KD protein and ESAT-6 protein were validated through ELISA,results suggested that both proteins were sensitive in TB serological diagnosis.Our study laid a foundation for the development of mycobacterium tuberculosis diagnostic kits.