【作者】 杨瑞丽；

【导师】 吴玉澄； 张耀洲；

【作者基本信息】 浙江大学 ， 特种经济动物饲养学， 2002， 博士

【摘要】 (一)家蚕／BmNPV表达载体系统(家蚕生物反应器)作为外源蛋白的低成本高效生产体系日益受到国内外的关注，数百种有用蛋白已在家蚕中获得成功的表达。外源蛋白基因在家蚕中的表达水平相对其他表达系统已很高，但仍远远低于野生型病毒的多角体蛋白在家蚕中的表达量。为了进一步提高外源蛋白在家蚕／BmNPV表达系统中的表达水平，我们以乙肝病毒表面抗原(PreS2+S)为研究对象，探讨了多角体基因序列对外源蛋白在家蚕中表达的作用及其作用机制，并对该基因高效表达的环境条件进行了优化。本研究通过PCR突变的方法，在HBsAg(PreS2+S)前S2序列的5’端融合了BmNPV多角体蛋白基因5’端的12个碱基，获得了融合乙肝表面抗原中蛋白基因(HBMp)。将克隆到的HBMp基因通过适当的酶切插入到转移载体质粒pBac-PAK8的多克隆位点中，获得重组转移载体质粒pBacPAK-HBMp。然后将重组转移载体质粒pBacPAK-HBMp与线性化的杆状病毒Bm-BacPAK6 DNA共转染家蚕培养细胞，二者在胞内发生同源重组，通过空斑筛选、DNA点杂交等手段鉴定出含有HBMp基因的重组病毒BmPAK-HBMp。用重组病毒BmPAK-HBMp和BmPAK-HBM[带有非融合乙肝表面抗原(PreS2+S)基因，为本实验构建]感染家蚕细胞及蛹，对两种病毒的表达产物用ELISA进行了跟踪检测，结果表明，感染BmPAK-HBMp的家蚕细胞及蛹中rHBsAg的表达量分别为3． 98μg／2×106细胞及46． 6μg／mL蛹血淋巴；感染BmPAK-HBM的家蚕细胞及蛹中rHBsAg表达量分别为2． 58μg／2×1O6细胞及26． 3μg／ml蛹血淋巴；前者与后者相比，HBsAg表达量分别提高了60％和80％。BmPAK-HBMp表达的融合M蛋白(PreS2+S)中，PreS2抗原性显著提高，与BmPAK-HBM表达的非融合蛋白相比，融合蛋白PreS2抗原性在细胞中约提高了8倍，蛹中约提高了1O倍。重组病毒BmPAK-HBMp在家蚕中表达时，检测到PreS2-Ag比HBsAg早1～2d达到最大值，重组病毒感染后的细胞，PreS2-Ag在第3d表达量最高，而HBsAg在 第sd表达量最高；BInP；AK－HBMP感染后的蛹，PreSZ个g的表达量在第sd达最大 值，而 H13sAs在第 6d达最大值。由此推测M蛋白（PreSZ＋S）的表达早于只含 S 区的小蛋白。另一方面，ELISA检测表明，PreSZ抗原性提高率约是 HBsAg的 14 倍，提示BmPAK－HB帅中多角体基因部分碱基序列的存在更有利于从PreSZ前的 ATG开始表达中蛋白全基因（PreSZ＋S）。 Western杂交结果表明，感染 BmPAK－HBMp和 BmPAK－HBM的家蚕蛹血淋巴中均 有 4种不同分子量的蛋白质和鼠抗人的一抗发生免疫反应，大小约分别为 31 kD、 30 kD、ZS kD、25 kD。推测31 kD处的条带为起始于PreSZ起始密码子ATG的表 达产物，同时具有PreS：Z和S抗原性，30 kD处的杂交条带为起始于S基因起始 密码子 ATG表达产物的糖基化形式，25 kD的条带可能是中蛋白的降解产物，而 28 kD的条带可能25 kD条带的糖基化形式。 对重组乙肝表面抗原（PreSZ＋S）基因在家蚕中高效表达所需环境条件的优 化结果表明，外源基因表达的宿主蚕品种对表达量的影响很大，rHBsAg在浙蕾X 春晓、丰一 X 54A及白王ZX新杭中的表达量最高，在 871X 872中的表达量最低， 不同品种间之间的表达水平可相差8倍，可见选择适当的表达宿主（蚕品种）可 以大幅度提高外源基因的表达产量。蛹接种后保育的温度对蛹的表达活性也有较 大的影响，保育在23OC·～26oC中蛹表达活性是保育在30C中的32倍，在20C中 的表达量下降1倍。 接种病毒的滴度及不同饲养季节对表达活性有一定的影响。较高的接种病毒 滴度能获的较高表达量的外源蛋白。病毒滴度为 IX 10丫fu时，rHBsAg可获得最 高表达活性，而低于 1＞ 10’pfU表达活性下降。春季蚕的表达量比秋季高 1倍左 右。我们还经一步探讨了新鲜蚕蛹（茧）低温贮藏对rHBsAg（PreSZ乃）表达 活性的影响。实验表明，4 C下贮存半个月对 rffesAg的表达活性没有太大的影 响；但冷藏时间超过半个月YHBSAg表达量明显降低。‘（二） 有研究表明，乙肝表面抗原小蛋白 u）和中蛋白基因（PreSZ6）在家蚕中 能获得高水平表达，并且其表达产物能聚集成22n。的颗粒。但家蚕表达的乙肝表 面抗原蛋白的兔疫原性至今在国内外尚未见报道。 11 为了探讨家蚕蛹表达的重组乙肝表面抗原（PreSZ＋S）的兔疫原性，本研究将重二 组病毒BmPAK十BM感染后的家蚕蛹以注射免疫的方式免疫SD鼠，结果发现家蚕蛹 表达的rHBsAg（Pr。，SZ＋S）能诱导鼠体产生较强的抗．HBs抗?更多还原

【Abstract】 (一)Foreign genes can be expressed effectively at low cost in silkworm-BmNPV expression vector system (Silkworm Bioreactor),which is widely noticed in the world. Numerous useful proteins have been expressed and the expression levels were higher in silkworm-BmNPV expression vector than that of others systems. However,the quantities of foreign gene products are far lower than that of the original polyhedra. In this study,we explored functional mechanism of polyhedron gene sequence on expression level of HBsAg(PreS2+S) genes and optimized the conditions for high-level expression in Silkworm-BmNPV expression vector system.A 12 bp sequence of the 5’ end from the polyhedrin protein gene of BmNPV was ligated to the 5’end of HBsAg( PreS2+S ) protein coding sequence by PCR. The fusion product coding for HBV surface antigen medium sized (HBMp) with 4-additional AA of BmNPV polyhedrin protein was obtained. The recombinant transfer vector pBacPAK-HBMp was constructed by insertion of the HBMp coding sequences into the multiple cloning site of transfer vector pBacPAK.8. BmN cell line was co-transfected with pBacPAK-HBMp plasmid and linearized baculovirus BacPAK6 DNA by Dosper agent. The homology of recombinant virus BmPAK-HBMp was obtained and identified by plaque assay and baculovirus contains the HBMp gene was confirmed through PCR and DNA dot blotting.The expressed rHBsAg was determined by ELISA after infecting Bm-N cells and pupae with recombinant virus BmPAK-HBMp and BmPAK-HBM (containing nonfusion HBV surface antigen medium sized). The results showed that expression amount of rHBsAg infected with BmPAK-HBMp reached 3.98ug/2xl06cells and 46.6ug/mL in BmN cells and in pupa hemolymph respectively. On contrary,expression amount ofrHBsAg reached 2.58ug/2106 cells and 26.3ug/mL respectively where has infected with BmPAK-HBM. Expression amount of rHBsAg in the former was increased 60% and 80% respectively than the latter. Antigenicity of PreS2 of fusion protein was increased 8 times and 10 times in BmN cells and in pupa hemolymph respectively than nonfusion HBsAg. The results also showed that kinetics of HBsAg and PreS2-Ag was different,the time of the high expression level of PreS2-Ag was earlier one or two days than that of HBsAg,and the efficiency increased of PreS2-Ag is about 13 times which is higher than that of HBsAg. These data implied that the expression of HBV medium protein (encoded by PreS2 gene and S gene) could be earlier then that of small protein (encoded only by S gene). The polyhedrin gene sequence in recombinant virus BmPAK-HBMp can be more benefit to enhancing the expression of the HBsAg(PreS2+S) from ATG initial codon at the 5’ ends of PreS2 gene.Pupal hemolymph infected with recombinant virus BmPAK-HBMp and BmPAK-HBM showed four protein bands of about 31,’30,28 and 25 kD in the Western blotting profiles,due to the two ATG initial codons at the 5’ ends of both PreS2 and S gene. This implied that the 31 kD initiated from ATG codon of PreS2 gene,the 30 kD encoded by the S gene was glycosylated form of S protein,the 25 kD initiated from ATG codon of the PreS2 gene was disassembled form of 31 kD and the 28 kD was a glycosylated form of25kD.Under optimal condition the expression level of foreign gene was considerably affected on hosts (silkworm race),the highest difference of expression level in various races can reach up to 8 times. Zhelei Chunxiao,Feng 1 54A and Baiyu Xinhang are the races of choice for expression of HBsAg (PreS2+S). The temperature at which the pupae were kept after treatment was greatly affected the expression level of foreign gene. Expression level in pupa is 32 times higher at 23C -26C than that at 30C,and decreased by 50% at 20C,The level of expression was influenced to some extent by the titre of virus used for infection and rearing season. Higher level of expression of HBsAg can be obtained from pupa infected with higher virus titre. The expression level was 1 time higher inspring than that of autumn. The expression level was decreased little in coco更多还原