| 李林林， 孙敏华， 董嘉文，等.表达番鸭细小病毒VP3基因重组鸡痘病毒转移载体的构建[J].广东农业科学,2015,42(3):134-139 |
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表达番鸭细小病毒VP3基因重组鸡痘病毒转移载体的构建 |
| Construction of recombinant fowlpox virus transfer vector expressing VP3 gene of muscovy duck parvovirus |
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| DOI： |
| 中文关键词: 番鸭细小病毒 VP3 基因 重组鸡痘病毒 |
| 英文关键词: muscovy duck parvovirus VP3
gene recombinant fowlpox virus |
| 基金项目: |
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| 摘要点击次数: 1589 |
| 全文下载次数: 805 |
| 中文摘要: |
| 根据 Genbank 上痘病毒 TK 基因序列分别设计左右同源臂引物 TKL-F/R 和 TKR-F/R，以 FPV 基
因组为模板，扩增左右同源重组臂（TKL、TKR）；PCR 连接左右同源臂，使中间带有多酶切位点 MCS，然后连入
pBluescript II SK（+）骨架质粒，获得的阳性质粒命名为 pTK；合成反向串联表达盒：p7.5-MCS 1-SV40-MCS 2-
P11，将表达盒插入质粒 pTK 的 MCS 位点，筛选获得阳性质粒 pTKS；在鸡痘病毒早期启动子 P7.5 后的 MCS1
处插入 MDPV VP3 基因，晚期启动子 P11 后的 MCS2 处插入 EGFP BGH pA，从而构建 MDPV VP3 基因重组
鸡痘病毒转移载体 pTKS-VP3-EGFP， 对构建好的转移载体进行酶切和测序鉴定。 将转移载体 pTKS-VP3-
EGFP 转染鸡痘病毒感染的鸡胚成纤维细胞（CEF）后，经 RT-PCR 和绿色荧光蛋白基因表达鉴定了 MDPV VP3
基因的表达，为进一步研制安全、高效的 MDPV 重组鸡痘病毒疫苗奠定了基础。 |
| 英文摘要: |
| In order to construct the recombinant fowlpox virus vaccine against muscovy duck parvovirus
(MDPV), 2 pairs of primers were designed amplifying two arms of the thymidine kinase (TK) gene (TK left and TK
right),according to genome of fowlpox virus FM strain on Genbank. Segment of TK left and TK right were linked by
overlap PCR, inserted a segment of multiple clone site (MCS). The TK left-MCS-TK right segment were inserted to
vector pBluescript II SK (+), and the abtained positive plasmid was named as pTK. The synthetized reverse tandem
expression cassettes p7.5-MCS 1-SV40-MCS 2-P11 were inserted to MCS of plasmid pTK, and the abtained positive
plasmid was named as pTKS. VP3 gene of MDPV was cloned into downstream of early promoter P7.5 and reporter
gene EGFP was cloned into downstream of late promoter P11 on plasmid pTKS. The recombinant fowlpox virus
transfer vector pTKS-VP3-EGFP were constructed and confirmed by digestion and sequencing. Plasmid pTKS-VP3-
EGFP was used to co -transfect into CEF with fowlpox virus vaccine strain. The expression of VP3
in the recombinant fowlpox virus was confirmed by RT-PCR and expression of green fluorescent protein.
This study provided useful information for development of a safe and effective recombinant FPV
vaccine against MDPV. |
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