摘要
乙型肝炎是危害全球人类健康的最主要的传染病之一，能够在全球广大地区流行。接种乙肝病毒疫苗已成为控制乙型肝炎发生的唯一经济有效的方法。如何使乙肝疫苗更加安全、经济、有效和更容易地推广使用是世界各国研究的主要方向。
随着基因工程技术和分子生物学研究的飞速发展，植物已开始被用作生产蛋白质、糖类和脂类等生物大分子的生物反应器。本试验以从genebank中查到的中国人群优势亚型adr亚型的乙肝病毒S基因序列为模板设计了正向引物5′-cgggatcc atggagaacacaacatcagga-3′和反向引物5′-cgagctc ttaaatgtatacccaaagaca-3′，以从乙肝患者血清中提取的DNA为模板进行了PCR扩增，获得了正确的S基因片段，经纯化、酶切，将此片段克隆到植物表达载体pBI121中，转化宿主菌大肠杆菌JM109；PCR和双酶切检测证明乙肝病毒表面抗原S基因已经插入到pBI121中，成功实现了在大肠杆菌细胞中的克隆；将此重组质粒用CaCl_2介导的热击法转化根癌农杆菌LBA4404，获得了阳性克隆，可以用于植物转化实验。同时本研究从愈伤组织再生和子叶外植体直接分化花芽两条途径对黄瓜组织培养条件进行了摸索，通过外植体类型、外植体苗龄和培养基中激素浓度配比几个方面对黄瓜愈伤组织的诱导进行了研究，认为外植体直接分化花芽的途径成苗时间短，再生率高，适合作为基因转化的受体系统，为进一步对黄瓜的遗传转化提供了依据。
本研究克隆的乙肝病毒表面抗原S基因来自于中国人群中的优势亚型adr亚型病毒，因此对于防治中国人群中广泛流行的乙型肝炎更具有针对性。将对HBV黄瓜疫苗的研制、改变传统免疫观念、改善免疫方法产生一定影响。
Hepatitis B is one of the most dangerous infectious diseases that do harm to people's health and it ravages in almost everywhere on the earth. Widely vaccination for hepatitis B has been the only economical and effective means to control the disease. And it is one of the most crucial tasks for us in the near future to find out how to make vaccine more safe, economical, effective and easy to application.
With the rapid development of molecular biology and gene engineering, plants can be used as the bioreactor for proteins, carbohydrates and lipids. Subtype adr is the prevailing subtype of HBV in Chinese people. We used the coding sequence of this subtype searched from genebank as the model of PCR reaction and designed a pair of primer: 5'-cgggatccatggagaacacaacatcagga-3' (Forward primer), 5'-cgagctcttaaatgtatac ccaaagaca-3' (Backward primer). The DNA prepared from the serum of HBV sufferers was amplified by PCR and the S gene of HBsAg was then obtained. After purified and digested by restricted enzymes, the S gene of HBsAg was successfully cloned into pBI121, which was a kind of plant expression vector. The recombinant plasmid was transformed into E. coli strain JM109 and then transformed into Agrobacterium tumefaciens LBA4404 by heat shock, which can be used for plant transformation. In the same time, we had an attempt in tissue culture of cucumber (Cucumis sativus L.) in two different ways and the effect of the explant tissue type, age of explant and hormone ratio in the medium on callus induction was investigated. We found that buds directly regenerated from cotyledon explants is suitable for plant transformation due to its high regenerating rate and quickly seedling, which established a basis for further study on the transformation of cucumber.
S gene of HBV subtype adr used in this research is the prevailing subtype in Chinese people, which make it more aiming in the precaution of HBV. This work can have certain impact on the development of cucumber-derived HBV vaccine and make a change in immunological concept and an improvement in the traditional immunological methods.
引文
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