【作者】 万家余；

【导师】 张玉静；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2005， 博士

【摘要】 我国属于甲、乙型肝炎高发区,主要靠接种甲、乙型肝炎疫苗加以预防,由于现有的疫苗生产成本高,接种次数多等特点,使得这些疫苗难以推广应用,特别在贫困地区,需要研制一种安全、高效、方便和经济等优点的甲、乙型肝炎双价疫苗。目前,利用减毒沙门氏菌作为载体的多价DNA 口服疫苗,具有生产、免疫方便等优良特点。本实验构建甲、乙型肝炎病毒的融合抗原基因SVPX,首先进行体外毕赤酵母细胞和CHO 细胞表达研究,结果表明,融合抗原基因在真核细胞里表达的蛋白具有甲、乙型肝炎抗原特性。构建含融合抗原基因的重组DNA 疫苗,以减毒沙门氏菌载体,进行小鼠口服免疫研究,结果表明,重组DNA 疫苗能够诱导机体产生针对两种抗原特异性体液和细胞免疫反应。同时,用IL-18 作为基因佐剂,能够进一步增强体液和细胞免疫的作用。本实验首次进行以减毒沙门氏菌为载体的甲、乙型肝炎口服二价DNA 疫苗的研究。更多还原

【Abstract】 People in the world infected with HBV had a total of 20 hundred millions, and there were a total of 6.9 hundred million people in china. People with HBsAg had a total of 3.5 hundred millions, and one thirth of people with HBsAg were in china.In the world every year people’s deaths had 750 thousands after they infected HBV, and there were 280 thousand people in china. People in china infected with HAV had a total of 9.7 hundred millions, and infectious rate had exceeded 80%. HB and HA largely harmed to people. At present there had not special therapy to HB and HA which were mostly prevented by vaccine.Though HB vaccine, HA vaccine or combined vaccine of HB and HA were important to prevent HB and HA, there was much to be treated.For example, recombinant vaccine, attenuated vaccine, inactivated vaccine were producted, trafficed, deposited and selled in low temperature and it was expensive.The vaccines were not popularized in poor region. , and many diseases were infected by injecting syringe.It was very important to develop a new double valence DNA vaccine of HBV and HAV, which was easily immunized, low charge, and good immune effect. DNA vaccine was immunized by many methods. Attenuated Salmonella typhimurium with DNA vaccine was easily immunized and economical. At the same time genetic background of Salmonella was clearly known and easily done by people. Because antibiotics in vaccine was not permitted by FDA of America, prokaryotic expression vector asd-balanced lethal system was constructed by Nakayama, which had no antibiotics. In our study, fusion gene of HBV and HAV antigen which was not expressed in prokaryotic expression vector was expressed in eukaryotic expression vector asd-pVAX1, which was constructed in the study and was complementary to E.coli X6212, Salmonella typhimuriumX3730 and Salmonella typhimuriumX4550. Hybrid gene SVPX was constructed by overlapping PCR with template of S gene of HBsAg and VPX gene of HAV complex multiepitope , and was then expressed in Pichia yeast and CHO host cells. Recombinant vectors asd-pVAX1-SVPX and pVAX1-SVPX were respectively transformed into Salmonella typhimurium X4550. Recombinant Salmonella typhimuriumX4550 with asd-pVAX1-SVPX and pVAX1-SVPX were immunized to mouse, then specific humoral and cellular immunity were detected. Hybrid gene SVPX whose DNA fragement was about 0.8kb, was constructed by overlapping PCR with template of S gene of HBsAg and VPX gene of HAV complex multiepitope. Specific primer was planned by us. Considering the special structure of two conformational epitopes, we inserted several flexible amino acids into two epitopes. A specific DNA fragment about 0.8kb was recovered. Ligation of the DNA with the vector pMD18-T was done and then transformed into competent host strain Escherichia coli JM109. Plasmids were isolated from recombinant clones by alkaline lysis, and PCR identification, restriction analysis and DNA sequencing were carried out to verify the recombinant plasmids pMD-SVPX. All results showed that the SVPX DNA was the exact fragment. According to Pichia yeast expression vector pPICZαC MCS and SVPX sequence, we planned two specific primers, and EcoR I & Kpn I were respectively belonged to upstream and downstream primer. A specific DNA fragment about 0.8kb was recovered. Ligation of the DNA with the vector pMD18-T was done and then transformed into competent host strain Escherichia coli JM109. Positive recombinant plasmid was sequenced ,and results showed that hybrid gene SVPX sequence was exact. The fusion gene SVPX DNA, cut from the recombinant pMD-SVPX with EcoR I & Kpn I, wasinserted into the yeast expression vector pPICZαC by the same digestion. The recombinant yeast expression plasmid was named as pPICZαC-SVPX. The recombinants were then transformed into competent Pichia yeast host cells GS115 by electroporation. The positive clones were screened by ZeocinTM resistance, and their genomic DNA were isolated and then amplified by PCR, using vector primers 5′AOX1 paired with 3′AOX1, and specific primers pP1 paired with pP2, to determine the integration of the interest gene into the Pichia genome. Most clones with ZeocinTM resistance were confirmed to be positive Pichia integrants by PCR amplification. When the recombinant Pichia strain was induced by adding of methaol to a final concentration of 0.5%, mRNA transcripted from the interest gene was detected by RT-PCR amplification, and the band of interest protein was found in the condensed supernatant by SDS-PAGE and Western blot. According to eukaryotic expression vector asd-pVAX1 and pVAX1 MCS and SVPX sequence, Specific primers were planned by us, and EcoR and Xho I were respectively belonged to upstream and downstream primer. In order to enhance the expression yields of the fusion antigen, a Kozak sequence was introduced into the designed fusion gene.A specific DNA fragment about 0.8kb was recovered.Ligation of the DNA with the vector pMD18-T was done and then transformed into competent host strain Escherichia coli JM109. Positive recombinant plasmid was sequenced ,and results showed that hybrid gene SVPX sequence was exact.The fusion gene SVPX DNA, cut from the recombinant pMD-SVPX with EcoR I & Xho I, was inserted into the eukaryotic expression vector asd-pVAX1 and pVAX1 by the same digestion. The recombinant eukaryotic expression plasmids were named as asd-pVAX1-SVPX and pVAX1-SVPX. CHO cells were transfected with recombinant plasmid asd-pVAX1-SPVX and pVAX1-SVPX by means of liposome respectively,and the fursion gene wasinstantaneously expressed. By RT-PCR amplification, ELISA assay and Western Blot, the fusion gene SVPX was expressed in transfected CHO cells successfully. Recombinant plasmids of asd-pVAX1-SPVX and pVAX1-SVPX were transformed into competent attenuated Salmonella typhimurium X3730 and X4550 in turn by electroporation. Recombinant attenuated Salmonella typhimurium X4550(asd-pVAX1-SPVX)and X4550(pVAX1-SPVX)were constructed and tested, and the results showed that the X4550(asd-pVAX1-SPVX)and X4550(pVAX1-SPVX)were stable, and could normally grow. Recombinant X4550(asd-pVAX1-SPVX)and X4550(pVAX1-SPVX)below 1×1010 cfu were safe when orally administered into BALB/c mouse. After recombinant X4550 ( asd-pVAX1-SPVX ) and X4550(pVAX1-SPVX)of 109cfu which is a safe dose were respectively orally administered into BALB/c mice for 3 times, the level of serum sepecific antibodies, cytotoxicity activity of specific CTL, the numbers of spleen T lymphocytes subgroups, and cytokine level of Th1 were examined.The results showed that the DNA vaccine could increase the percentage of the T cell subgroups , elicit serum specific antibody and specific cytotoxicity activity of CTL to HBsAg and HAAg. The immunized mice could product Th1 cytokine. Recombinant X4550(asd-pVAX1-IL-18) and X4550( pVAX1-IL-18) were constructed in our sudy. Combined group of X4550(asd-pVAX1-SPVX)& X4550(asd-pVAX1-IL-18), and X4550(asd-pVAX1-SPVX)& X4550(asd-pVAX1-IL-18)were orally adiminstered into BALB/c mice. At the same time there were control groups of X4550(asd-pVAX1-SPVX)and X4550(pVAX1-SPVX)with 109 cfu of Each recombinant X4550 was orally administered into BALB/c mice for 3 times.The level of serum sepecific antibodies, cytotoxicity activity of specific CTL, the numbers of spleen T lymphocytes subgroups, and cytokine level of Th1 were examined.The results showed that the percentage of the T cell subgroups , level of sera specific更多还原