| Hepatitis B is an infectious disease caused by hepatitis B virus (HBV). HBV is responsible to acute and chronic hepatitis, as well as hepatocirrhosis, liver cancer. Hepatitis B vaccine inoculation is the most effective way to prevent the disease. The hepatitis B vaccine presently used in China comprises major S protein only. Its disadvantages are: (1) 5%-10% of the recipients raise only little or even no response; (2) three inoculations are needed to obtain essential antibody level; (3) it is used only in prevention, but not therapy. So to prevent quickly and enhance protection still are the problem that a novel Hepatitis B vaccine needs solve. Nowadays researchers found that pre-S protein, with high antigenisity, can enhance the immunoresponse of S protein; production of pre-S2 antibody is earlier than S antibody, it plays an important role on early prevention. And pre-S2 can adhere to hepatocytes via poly human serum albumin (PHSA). Therefore vaccine containing pre-S2 can prevent HBV from infecting liver cells by competitive joining. So it may cure hepatitis. More and more date show that a novel hepatitis B vaccine with pre-S can overcome the disadvantage of only S-protein vaccine.In terms of vaccine designing strategy, the antigen molecule should be large, the formed polymer or particle is more, effect of immunogenicity is better. But in genetic engineering, the product molecule should be small to obtain high expression and to decrease the side effects. So it is necessary to consider the characteristic of vaccine and genetic engineering products on design genetic engineering vaccine.The topic leads in a new concept of epitope -based vaccine design, reasonably retains the whole S gene, chooses 26 amino acids sequence of pre-S2 (120-146) that contains rich T and B cell epitope, removes 29 amino acids unnecessary sequence of pre-S2 and makes the size of recombinant protein suitable and express easily. The designed vaccine by aboved method not only retains necessary antigen epitope of antigen stimulates, but also reduces influence on protein secretion because of the expressed large molecule protein. The preliminary results showed the vaccine had expressed S2S protein, the expression level and immunity effect surpassed current S vaccine.In the study, the whole S gene adding the epitope gene of pre-S2 (120-146) was cloned into a eukaryotic expression plasmid pcs and the recombinant plasmid pcs-S2S was constructed by the recombinant DNA technology . the plasmid pcs-S2S was transformed into CHO dhfr–cell successfully, totally screened 2400 cells, obtained recombinant CHO cell A7 strain. The A7 strain was subcultured for 50 passages and change medium for 30 times continuously, It is established that the stability of preS2S protein was good. The results of S and S2 proteins detected in CHO cells A7 strain were strong positive by DNA bot hybridization; expressed gene was detected by PCR, then sequenced, the length of S gene is 678 bp, the length of preS2 gene is 78 bp, the results of SDS-PAGE and sequencing were correct. It was verified that A7 strain cell contained S and S2 gene. Expressed protein preS2S was identified By SDS-PAGE and Western blot.The genetic engineering products in Europe and America are expressed mainly by CHO cell, about 40%~70%, there are 22 kinds products in America which has been authorized to go on the market, but only two kinds in China. This is major gap between China and Europe and America on Biological drugs manufacture. This topic chose CHO cell expression system, we tried to overcome many technique problems in transfection and screening, groped and solved the problem of clone and amplification of monoplast and successfully obtained recombinant CHO cell A7 strain that can express recombinant protein. It is a benefic attempt on CHO cell expression system.In the past antibody level was used to estimate a vaccine. But present research showed that cellular immunity plays an important role in those diseases caused by intracellular infection, e.g. hepatitis B. At present, vaccines for person using are still aluminium adjuvant vaccine primarily, we choose aluminium adjuvant to prepare S2S vaccine. In addition, on the basic of another study (patent), we use BCG to prepare S2S vaccine, which obtained better effect on humoral immunity and cell immunity.S2S-aluminium adjuvant vaccine was detected by RIA method, Immunity efficacy of S2S vaccine (relative efficacy were 4.1, 2.0, 1.97) was higher than S vaccine (relative efficacy was 1.57). Anti-HBs and anti-preS2 was detected by ELISA method, anti- HBs induced by S2S vaccine was higher than S vaccine, p<0.05, it showed significant difference. Although the level of anti-preS2 induced by S2S vaccine was higher than S vaccine, p >0.05, there was no significant difference. what is more, preS2 antibody induced by S2S vaccine reaches the highest level in second week, it is significant on early prevention.The humoral immunity of S2S-BCG vaccine was higher than S2S-aluminium adjuvant vaccine , there was no statistical difference, but cell immunity of S2S-BCG vaccine was higher than S2S-aluminium adjuvant vaccine, it showed significant difference. IL-2, lymphocyte stimulation indices and CD4+/CD8+ value of S2S-BCG vaccine were obviously higher than S2S-aluminium adjuvant vaccine. BCG can induce humoral immunity and cell immunity, but aluminium adjuvant has not this function, it recruits the deficient that current Hepatitis B vaccine only induces humoral immunity, the novel Hepatitis B recombination vaccine has potential application value on prevention and therapy Hepatitis B.In a word, It is shown to be good prospect for study of recombinant pre-S2S hepatitis B vaccine produced by CHO cells on enhancing immunogenicity and inducing cellular immunity. It can lay a foundation for further developing new type hepatitis B vaccine for prevention and therapy. |
