Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (24): 5125-5134.doi: 10.3864/j.issn.0578-1752.2020.24.015

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles    

Optimized Promoter Regulating of Duck Tembusu Virus E Protein Expression Delivered by a Vectored Duck Enteritis Virus in vitro

CHEN Liu(),NI Zheng,YU Bin,HUA JiongGang,YE WeiCheng,YUN Tao,LIU KeShu,ZHU YinChu,ZHANG Cun()   

  1. Institute of Animal Husbandry and Veterinary Medicine, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021
  • Received:2020-01-17 Accepted:2020-07-29 Online:2020-12-16 Published:2020-12-28
  • Contact: Cun ZHANG E-mail:haoliuzi@126.com;zhangcun@aliyun.com

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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

Table 1

Primers used in this study"

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

Fig. 1

Analysis of recombinant BAC mutants by Pst I digestion 1: pDEV-EF1; 2: pDEV-Es; 3: pDEV-kan. pRSV. Es; 4: pDEV-pRSV. Es; 5: pDEV-kan.pSV40. Es; 6: pDEV-pSV40. Es; 7: pDEV-kan.pCAG. Es; 8: pDEV-pCAG. Es; 9: pDEV-kan.p1.8k(MDV). Es; 10: pDEV-p1.8k (MDV). Es; 11: pDEV-kan.pgB(MDV). Es; 12: pDEV-pgB(MDV). Es"

Fig. 2

Analysis of recombinant BAC mutants by PCR amplification"

Fig. 3

The rescued viruses (100×)"

Fig. 4

Plaque area measurement of recombinant viruses on CEFs"

Fig. 5

Western blotting analysis of Es protein expressed in recombinant virus-infected CEFs 1: rDEV-Es; 2: rDEV-pSV40-Es; 3: rDEV-pCAG-Es; 4: rDEV-pRSV-Es; 5: rDEV-p1.8(MDV) -Es; 6: rDEV-pgB(MDV)-Es; M: EasySee Western Marker (90, 75, 60,40, 25 kD); 7: rDEV-EF1"

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