重组鸭瘟病毒载体中筛选高效表达鸭坦布苏病毒E蛋白启动子

doi:10.3864/j.issn.0578-1752.2020.24.015

摘要/Abstract

摘要：

【背景】鸭瘟和鸭坦布苏病毒是鸭的两种重要传染病,鸭瘟属于疱疹病毒科,具有开发成病毒载体的优势。为了优化鸭坦布苏病毒E基因在重组鸭瘟病毒载体中的表达,之前探讨了不同形式鸭坦布苏病毒E蛋白在重组鸭瘟病毒载体中的表达,发现以鸭为宿主进行密码子优化的E基因C端截短形式（E451-dk,简称为Es）表达量最高。【目的】探讨不同启动子对Es 在重组鸭瘟病毒载体中表达的影响,为鸭瘟病毒—坦布苏病毒二联苗的研制奠定基础。【方法】将pCAG、 pSV40、pRSV、p1.8k(MDV)和pgB(MDV)启动子通过常规基因克隆的方法替换转移载体pEP-BGH-Es中的pCMV启动子,构建不同启动子调控Es表达的重组表达框pro-Es-BGH-pA。在鸭瘟病毒（DEV）疫苗株细菌人工染色体克隆pDEV-EF1的基础上,将5个重组表达框分别通过“Red E/T两步重组”克隆至pDEV-EF1突变体的US7和US8基因之间,构建了携带不同启动子调控的Es突变体克隆pDEV-pro-Es。用磷酸钙法转染鸡胚成纤维细胞（CEFs）拯救获得相应重组病毒rDEV-pro-Es,并对重组病毒感染细胞蚀斑大小和Es蛋白表达情况进行测定。【结果】将重组突变体克隆转染细胞拯救获得了5株重组病毒rDEV-pro-Es。Western blotting分析表明外源蛋白Es在 pRSV调控下表达量最高,其表达量较rDEV-Es提高了169.12%。【结论】完成了Es在重组鸭瘟病毒载体中高效表达启动子的筛选,获得了一种调控Es高效表达的启动子pRSV。同时也获得了一株高效表达鸭坦布苏病毒外源基因Es的重组鸭瘟病毒rDEV-pRSV-Es。

关键词: 鸭瘟病毒, 鸭坦布苏病毒, E蛋白, 细菌人工染色体, 病毒载体, 启动子

Abstract:

【Background】 Duck enteritis virus (DEV) and duck Tembusu virus (DTMUV) are considered to be two of the important viruses that infected ducks. DEV is classified into the family Herpesviridae, which has the characterization of live viral vector. 【Objective】In our previous study, a recombinant DEV delivering optimized DTMUV E451 gene (E451-dk) referring to duck’s codon usage bias has been selected. In this study, the promoter regulating E451-dk (Es in short) in rDEV-EF1 was also evaluated for enhancing E451-dk expression level. 【Method】 The transfer vector pEP-BGH-pro-Es were constructed by separately substituted pCMV (cytomegalovirus major immediate-early promoter) on the vector pEP-BGH-Es with pCAG (human cytomegalovirus enhancer and chicken-actin promoter), pSV40 (the simian virus 40 promotor), pRSV(Rous sarcoma virus (RSV) promoter), pgB（MDV）(marek's disease virus (MDV) gB gene promoter) and p1.8k（MDV）(MDV 1.8k gene promoter). The recombinant DEV BAC clone pDEV-pro-Es carrying pro-Es genes were generated by two-step Red E/T recombination in E. coli. pDEV-pro-Es were constructed by inserting pro-Es expression cassette between DEV US7 and US8 genes on the infectious clone of DEV (pDEV-EF1). The recombinant virus rDEV-pro-Es (rDEV-pCAG-Es, rDEV-pSV40-Es, rDEV-pRSV-Es, rDEV-pgB(MDV)-Es and rDEV-p1.8k(MDV)-Es) were rescued from chicken embryo fibroblasts (CEFs) by calcium phosphate precipitation. The plaque size and expression of DTMUV Es in recombinant virus-infected CEFs were analyzed. 【Result】 All viruses were successfully rescued from CEFs. Western blot analysis showed that the expression level of Es in rDEV-pRSV-Es -infected cells was increased 169.12% compared to that of rDEV-Es -infected cells. 【Conclusion】pRSV was the highest effective promoter chosen in this study which regulating Es expression on recombinant DEV genome backbone. These studies laid a foundation for developing bivalent vaccine controlling DEV and DTMUV infection.

Key words: duck enteritis virus, duck Tembusu virus, E protein, bacterial artificial chromosome, viral vector, promoter

陈柳,倪征,余斌,华炯钢,叶伟成,云涛,刘可姝,朱寅初,张存. 重组鸭瘟病毒载体中筛选高效表达鸭坦布苏病毒E蛋白启动子[J]. 中国农业科学, 2020, 53(24): 5125-5134.

CHEN Liu,NI Zheng,YU Bin,HUA JiongGang,YE WeiCheng,YUN Tao,LIU KeShu,ZHU YinChu,ZHANG Cun. Optimized Promoter Regulating of Duck Tembusu Virus E Protein Expression Delivered by a Vectored Duck Enteritis Virus in vitro[J]. Scientia Agricultura Sinica, 2020, 53(24): 5125-5134.

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引物名称 Primer	序列 Sequence	引入位点 Sequence introduced
pCAG(MluI+)	5′-cgACGCGTTAGTTATTAATAGTAATCAATTACG-3′	Mlu I
PCAG(NheI-)	5′-ctaGCTAGCGCCGCCGGTCACACGCCAGAAGCC-3′	Nhe I
pRSV(BglII+)	5′-gaAGATCTCTGCTCCCTGCTTGTGTGTTG-3′	Bgl II
pRSV(NheI-)	5′-ctaGCTAGCGTGCACACCAATGTGGTGAATG-3′	Nhe I
pSV40(MluI+)	5′-cgACGCGTCTGTGGAATGTGTGTCAGTTAGG-3′	Mlu I
pSV40(NheI-)	5′-ctaGCTAGCCGAAAATGGATATACAAGCTCCCGG-3′	Nhe I
F(MDV p1.8k BglII+)	5′-gaAGATCTTCGAGGCCACAAGAAATTAC-3′	Bgl II
R(MDV p1.8k NheI-)	5′-ctaGCTAGCGAGCATCGCGAAGAGAGAAG-3′	Nhe I
F(MDV gB BglII+)	5'-cgAGATCTCAAGTCTCACTCACAAATTTTTTC-3′	Bgl II
R(MDV gB NheI-)	5′-ctaGCTAGCAGTGAGATGATCTTAATGATGC-3′	Nhe I
pDEV vac-in-s(p1.8k,pgB)	5′-TACTAATTTAAGTGTGCAGCCTGGTTAACTGTATTATGCGCGGAGTGACGTCGACGGATCGGG-3′
pDEV vac-in-s	5′-TACTAATTTAAGTGTGCAGCCTGGTTAACTGTATTATGCGCGGAGCGATGTACGGGCCAGATA-3′
pDEV vac-in-as	5′- TCCGTAGTCTGGCCGGCAGTATGTTGGTGTTTAGTACTCCAAACCCA TAGAGCCCACCGCATCCCC-3′
JD-F	5′-CTACCACAAGCGTCATCAACCA-3′
JD-R	5′-TGTCCATTACCAAATCCGAAAA-3′
DEV-tk-F	5′- GCTTCCCAGCAGCTCGTT-3′
DEV-tk-R	5′- TCTCGTACTTCAGCGGCACA-3′
Dev UL44(BamHI+)	5′-cgGGATCCATGGGGCCATTAGTGATGGTTG-3′	BamH I
Dev UL44 (XhoI-)	5′-ccgCTCGAGTCAAATAATATTGTCTGCTTTATC-3′	XhoI