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| 白斑综合症病毒(WSSV)囊膜蛋白VP28基因的克隆及在蓝藻中表达载体的构建 | |
| 引用本文： | 张春莉,施定基,黄倢,张海霞,彭国宏.白斑综合症病毒(WSSV)囊膜蛋白VP28基因的克隆及在蓝藻中表达载体的构建[J].海洋科学,2003,27(2):72-76. |
| 作者姓名： | 张春莉 施定基 黄倢 张海霞 彭国宏 |
| 作者单位： | 1. 中国科学院海洋研究所,青岛,266071;中国科学院植物研究所,北京,100093;中国水产科学研究院黄海水产研究所,青岛,266071;中国科学院研究生院,北京,100039 2. 中国科学院植物研究所,北京,100093 3. 中国水产科学研究院黄海水产研究所,青岛,266071 4. 中国科学院海洋研究所,青岛,266071 |
| 基金项目： | 国家重点基础研究发展规划项目G1999012002号“对虾病毒病的分子与细胞学机理”资助。 |
| 摘 要： | 用蔗糖密度梯度离心法分离纯化白斑综合症病毒（WSSV）。设计并合成引物，提取WSSV中国株的基因组DNA作为模板，通过PCR，扩增克隆出VP28基因，利用BamHI和EcoRI切点将VP28基因插入克隆载体pUC19 的多克隆位点上，得到VP28基因的重组克隆质粒，对其进行双向DNA测序。测序结果表明，该基因含有615个核苷酸，与GenBank中已有不同来源的WSSV的序列片段同源性为100％。利用BamHI切点将VP28的基因插入到穿梭表达载体启动子PpsbA的下游，EcoRI酶切鉴定，得到正向连接的可在蓝藻表达的重组穿梭表达载体，命名为pRL-VP28。 |
| 关 键 词： | 白斑综合症病毒 囊膜蛋白VP28 蓝藻 表达载体 构建 对虾 基因克隆 |
| 文章编号： | 1000-3096(2003)02-0072-05 |
| 收稿时间： | 7/8/2002 12:00:00 AM |
| 修稿时间： | 2002年7月8日 |
CLONE OF ENVELOPE PROTEIN VP28 GENE OF WHITE SPOT SYNDROME VIRUS (WSSV) AND EXPRESSION VECTOR CONSTRUCTION FOR CYANOBACTERIA |
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| Abstract: | This paper deals with the molecular mechanism of the infection of Penaeus chinensis by white spot syndrome virus. A stain of WSSV was isolated and purified from P. chinensis with sucrose gradient centrifugation. Protein VP28 was identified by SDS-PAGE. Genome DNA of WSSV was extracted. The primer was designed and synthesized according to the reported sequence in the GenBank. The gene of VP28 protein was cloned from Genome DNA with PCR amplification. The product of PCR digested by restriction enzyme BamHI and EcoRI was constructed on the clone vector pUC19. The recombinant vector was identified with double digestion of BamHI and EcoRI. The gene was sequenced and the results showed that the VP28 gene contains 615 nucleotides encoding 204 amino acids and 100% identity with WSSV sequences reported in GenBank. The clone vector was named as pUC-VP28. Then, plasmid pUC-VP28 digested by BamHI (3.3 kb) was ligased with pRL-489 digested by BamHI (9.0 kb). Recombinant was selected with double antibiotics and was identified with digestion with BamHI. The VP28 gene inserted at the same site at downstream of the promoter PpsbA moves towards different directions. There are two EcoRI sites: one is at upstream of the promoter PpsbA and the other located at the 3' end of vp28 gene. The recombinant was digested with EcoRI and 1.0kb was seen in the gel of 0.6%. The result showed that the vp28 gene was located at the downstream of PpsbA. The recombinant shuttle expression vector for cyanobacteria was named as pRL-VP28. It was the key step for expression of VP28 protein in cyanobacteria. |
| Keywords: | White spot syndrome virus (WSSV) Envelope protein VP28 protein Gene clone Shuttle expression vector |
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