Expression And Optimization Of The Fragment Including S Gene Whole Antigenic Sites Of Swine Transmissible Gastroenteritis Coronavirus

Transmissible gastroenteritis of swine (TGE) which is caused by transmissible gastroenteritis virus of swine (TGEV) is an acute, highly prevalent enteric infectious disease. TGEV have four major structural proteins. The major antigenic sites of TGEV involved in the induction of virus neutralizing antibodies are located in the globular portion of the spike (S) protein. The S protein of TGEV has four major antigenic sites, which can induce immune protecting effects as same as the whole S protein does. So it is vital to get a DNA fragment including S gene whole antigenic sites in swine transmissible gastroenteritis coronavirus in order to prevent and treat TGE.In this research, the DNA fragment including S gene whole antigenic sites was cloned and inserted into pET-28a vector. The recombinant plasmid transformed into E.coli strain BL21 (DE3) to induce the expression of S protein with IPTG. SDS-PAGE and Western-blot indicated that the expression of target protein was the same as the expected results. Through auto-induction expression system, the amount of target protein was improved significantly. The results of research are as following:1. A DNA fragment including S gene whole antigenic sites in swine transmissible gastroenteritis coronavirus which was 2.1 kb was amplified by PCR. The recombinant expression plasmid pET-S1 was constructed by inserting target gene into pET-28a vector.2. The recombinant plasmid pET-S1 was transformed into E.coli strain BL21 (DE3) to induce the expression of target protein under 37℃, 30℃and 28℃. Stability of cultures was tested by plating on agar plates after 105-fold dilution. The results indicated that 60 % and 30 % E.coli strain lost pET-S1 when strain was induced under 37℃and 30℃, respectively. The recombinant plasmid can maintain stability under 28℃.3. The recombinant plasmid pET-S1 was transformed into E.coli strain BL21 (DE3) to induce the expression of target protein under 28℃. The result of SDS-PAGE and Western-blot indicated that the target protein was about 76 ku which is the same as the expected length and maintained antigenicity of TGEV. The highest amount of target protein accounted for 25.9 % of total bacteria protein. 3.30 % target protein can be detected in non-induced E.coli cultures.4. The results of optical density at 600 nm (OD600) of non-induced cultures and induced cultures demonstrated that the saturating density of non-induced cultures doubled the induced cultures to OD600~6.19.5. Through auto-inducting expression system, the recombinant plasmid transformed into E.coli strain BL21 (DE3) which was cultured in the changed culture medium and condition. the highest amount of target protein accounted for 45.1 % of the total bacteria protein.6. In IPTG-induction systerm, the highest OD600 of cultures was 2.63, and cultures had a trend of turning to alkaline. While in auto-inducing systerm, the highest OD600 of cultures was 12.65, the cultures can maintain neutral.