Cloning And Expression Of Recombinant Ag85A DNA

PrefaceAg85 complex is a family of exported fibronectin binding protein and recent studies have shown these proteins to have mycolyl transferase activity and also in cell wall synthesis. The mycolyl transferase activity is required for the biogenesis of trehalose dimycolate, a dominant structure necessary for maintaining cell wall integrity.The protein components that make up the Ag85 complex include Ag85A, Ag85B and Ag85C and are prominently represented by Ag85A and Ag85B. Ag85A is expressed in culture filtrate from surface grown Mycobacterium tuberculosis and Mycobacterium bovis. The use of native mycobacterium protein in serodiagnosis, fundamental immunological studies and subunit vaccination is hindered by difficulties in growing the organism and purifying the antigens. Antigen aggregation, degradation in cell extracts and particularly slow and fastidious growth are the main problems associated with purification of antigen from myco-bacterial cultures. The interest in recombinant Ag85A has been due to the discovery from animal studies that it has significant effect on cellular and humoral immunity and could serve as candidate for possible vaccine against tuberculosis and other infective diseases. The ability to target antigen presenting cells with vector encoding desired antigens holds the promise of potent prophylactic and therapeutic vaccines for infectious diseases and cancer.The objective of this research was to clone Antigen 85A into pTrcHisB and the recombinant protein expressed in E. coli TOP 10. pTrcHisB vector has a high expression capacity in E. coli. There is no recorded article in China on the cloning and expression of Recombinant Ag85A using pTrcHisB as the expression vector. The pTrcHisB vector has high expression capacity in E. coli. The idea behind this research is to show that the Ag 85A DNA obtained from Pasteur Re-search Institute, Belgium is a viable DNA for further research in vaccine production. This research made use of the simple expression system of a prokaryotic bacteria like E. coli in demonstrating that the recombinant protein of Ag 85A could be expressed. This is a step forward towards producing DNA vaccine from the Ag85A DNA.Method1. Extraction of Ag85A DNA: This involved preliminary culture of the Salmonella typhimurium in LB media to obtain pure colonies. Extraction of the Ag85A - VIJns. tPA plasmid from the bacteria was done using pEZ Spin Column Plasmid DNA Minipreps Kit. The extracted plasmid was confirmed by electrophoresis on 0. 8% Agarose gel. The Ag85A - VIJns. tPA plasmid was digested using BgUI to release the Ag85A DNA fragments and this was purified using QIAquick gel extraction kit.2. Digestion of pTrcHisB vector*. The pTrcHisB was digested at with BgUI. At the end of the digestion, the product was treated with Alkaline Phosphatase to dephosphorylate the 3' and 5' ends. This was done to prevent the two cohesive ends from rejoining. Purification of enzyme digested vector was done using Qia-quick Gel Extraction Kit. Purified samples were stored at -20TI until use.3. Ligation of Ag 85 A DNA to BglII digested pTrcHisB : Ligation of the Ag 85 A DNA to the pTrcHisB was done according to the protocol for the T4DNA ligation kit. Extraction and purification of the ligated product was done using QIAquick gel extraction kit. Agarose Gel Electrophoresis was run to confirm successful ligation.4. Transformation of Recombinant Ag 85A DNA into E. coli TOP10- The transformation of the competent E. coli TOP 10 was done according to the procedure provided by Invitrogen. The transformation technique used was the CaCl2 method. The E. coli TOP10 cells were treated with ice - cold 50mM CaCl2 lOjxl of 10ng/l of transforming Antigen 85A DNA was added to appropriate aliquots of 100 l CaCl2 - treated E. coli TOP10. Transformed E. coli TOP10 cells were plated out on to SOB culture plate containing Ampicillin (50Lg/ml) and theplates incubated overnight at 37. After overnight incubation, the plates were examined for positive transformation.5. Test for Protein Expression: Overnight colonies selected (ampicillin) on the plates were subcultured on to LB liquid for amplification. The recombinant plasmid was extracted using pEZ Spin Column Plasmid DNA Minipreps Kit. Agarose Gel Electrophoresis of extracted plasmid was done to confirm successful transformation. SDS - PAGE was run to confirm expressed protein. Further confirmation of the Recombinant Ag 85 A protein was done by Western blotting using HYT27 monoclonal antibody. Anti - Rabbit polyclonal antibody conjugated to Horse Radish Peroxidase was used as the second antibody.Result1. Extraction of Ag85A DNA: The Ag85A was successfully extracted from the Ag85A - Vljns. tPA by Bglll digestion and after electrophoresis on 0. 8% Agarose gel, the correct band of 1014bp was obtained.2. Ligation of Ag 85 A DNA to Bglll digested pTrcHisB: The ligation of the 1014bp Ag85A to the Bglll digested 4404bp pTrcHisB produced a recombinant Ag85A -pTrcHisB with 5418bp confirmed by electrophoresis on 0.8% Agarose gel.3. Transformation of Recombinant Ag 85A DNA into E. coli TOP10: The sample plates which had positive transformation showed scanty growth of the E. coli TOP10. The Negative Control showed no bacteria growth even after 48 hours of incubation at 37X1. The Ampicillin Control however showed heavy growth of bacteria after overnight incubation.4. Test for Protein Expression; The SDS - PAGE of the repombinant Ag85A produced a band of about 35kDa and this corresponded with the result of Huygen et al (1997). For the western blotting, after reaction with the monoclonal and secondary antibodies, a band was obtained and this corresponded with the result of Lakey et al (2000).