| Tuberculosis (TB) is a chronic respiratory infection caused by the causative agent of Mycobacterium tuberculosis (MTB). It has been estimated that one-third of the world's population is infected with Mycobacterium. An estimated 10 million new cases of TB and 2 million deaths are reported every year, making TB remains one of the most leading infectious diseases of humans. TB represents a major problem for public health worldwide. The count of TB patents in China is the second in the world, approximately five hundred million persons, roughly 50 million develop active tuberculosis and about 250 thousand do not survive per year of these individuals. People died in TB are nearly two times as died in other infectious diseases. BCG is widely administered worldwide, but it provides protection against TB varies from 0% to 80%. Currently, the diagnosed method is still poor sensitivity. With the drug-resistent strains spread, human immunodeficiency virus co-infection and people migrated frequently, it is urgent to invent new methods to pretent TB.Ag85B and ESAT6 are both crucial protective antigens in the early culture filtrate protein(CFP) which is released in the early logarithmic growth stage. They can induce human protective immunity. Resuscitation promoting factor was first found in Micrococcus luteus, which can stimulated growth of dormant M.luteus.AIM To construct the prokaryotic vectors, then express and purify the proteins Ag85B,Ag85B-ESAT6 and ESAT6-RPFD. Produce the monoclonal antibody against the protein Ag85B.METHODS AND RESULTS1. Genes of ag85B, esat6, rpfD were amplified with specific primers designed from the MTB H37RV strain by PCR. Inserted the PCR products into pMD19-T vector which were match tbe sequences supported by GenBank. Then target genes were inserted into pProEXHTb and expressed in DH5αindeced with IPTG. The SDS-PAGE showed that there were specific proteins expressed at 32 kD, 43 kD, 30 kD. These proteins were identified by Western-Blot. Puried these proteins via Ni-NTA purification system under denaturing condition and measured the concentration. The results respectively were 1.16 mg/mL;0.78 mg/mL;0.54 mg/mL. We could get 25.52 mg, 17.16 mg and 23.76 mg of purified protein from 200 mL culture respectively and purified protein yield were 4.4%, 3% and 4.1%.2. Resuspend 100μg purified protein in the aqueous solution. Then transfer the resuspended antigen to mix with commensurate Freund's incomplete adjuvant and make emulsion of antigen-adjuvant and agitate to dissolve the adjuvant. Injected the solution made subcutaneously with 100μg protein a single. Two weeks later, boost immunization is followed. After several boost immunization, tested the serum titer against antigen, chose the positive immunization results for future fusion. Kill the mouse and remove the spleen, mix 1:10 splenocytes and myeloma. Then added fused-cell suspension into 96-well culture plates layered with feeder cells made the previous day. Refresh culture medium in half at 72 h post-fusion, replace HAT with HT to facilitate newly-hybrid cells to proliferate. Three mAb hybridoma cell lines were obtained. All of these are IgG1 subclass. Inject the hybridoma cell into peritoneal cavity to produce ascites, then purified the ascites through ASA solution. The mAb were identified by Western Blot, IFA and ELISA. Specificity test results of mAbs showed that they acted with MTB instead of Staphylococcus aureus cell lysate and E.coli cell lysate.
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