Cloning And Expression Of Mutant Hepatitis B Surface Antigen In Pichia Pastoris And Their Recognition By Commercial Detection Assay

Hepatitis B virus (HBV) infection is a serious healthcare problem . Infection can be controlled successfully by such measures as universal vaccination, passive immunization, and the recent anti-viral treatment options . However, it is a long-term and hard task to control the disease. With the deep research to HBV, some new problems have emerged. Mutations in the hepatitis B surface antigen (HBsAg) gene occured as a result of vaccine escape mutants, anti-HBs immunotherapy, or in chronic hepatitis B viral infection. These mutations may present a new challenge to immunoassay detection. A lot of articles reported that HBsAg diagnosis kits failed to detect these mutations. But there were few reports about the epidemiology of HBV mutant and the effection of mutation to HBsAg diagnosis kits.We have four plasmids ligating T-vector, one containing wild HBV S gene and the others containing three mutants (T126N, D144A, G145R). Using the four plamid as templet, we operated PCR to get aim genes, then ligated the aim genes to the plasmids pPICZA and pPICZα.We have constructed six yeast expression plasmids. Reading frame and nucleoside sequence of them were correct after sequencing .All these expression plasmids were linearized with Sal I .These linearized plasmids were transformed into the host cell of GS115 with electroportaion method. After screening with Zeocine and inducing with methanol, we got several high-expression transforms. The results of ELISA and Western Blot proved out that the expression products were HBsAg.The second part of my project is that evaluating the HBsAg diagnosis kits' ability to detect the mutants by using many mutants HBsAg. Many factors can effect the results of ELISA, so we firstly normalized our operation using a serial dilution of HBsAg positive serum. And we regarded this serum as standard to control afterward operation.We have three kinds of mutant HBsAg, from yeast expression system, CHO expression system and mouse L cell expression system, respectively. These mutant antigens were diluted in normal human serum previously screened to be HBsAg and anti-HBs negative and detected by different diagnosis kits .The results of ELISA showed that the products of different expression system got same conclude, that is, all 4 domestic assays may lack sensitivity to detect the different mutant antigen. The G145R mutant and other mutants relating to G145R mutants were the most challenging to detect by domestic HBsAg diagnosis kits. The T118K/P120Q, Q129R/M133T mutants have some effect on domestic assays. And T126N, D144A mutants have no effect on domestic assay nearly. At last, we used a serum containing 1/4 G145R mutant to investigate the effect of diagnosis kits. The results showed that the sensitivity of domestic assays decreased.