PMID- 23547375
OWN - NLM
STAT- MEDLINE
DCOM- 20130613
LR  - 20161021
IS  - 1000-8721 (Print)
IS  - 1000-8721 (Linking)
VI  - 29
IP  - 1
DP  - 2013 Jan
TI  - [Study on using NSP2 protein of porcine reproductive and respiratory syndrome 
      virus (HuN4-F112) to express E2 neutralizing epitope of classical swine fever 
      virus].
PG  - 17-25
AB  - Establishment of recombinant porcine reproductive and respiratory syndrome virus 
      (PRRSV) with co-expression E2 Epitope of Classical Swine Fever virus (CSFV) is a 
      crucial step to develop a genetic engineered vaccine against PRRSV and CSFV. 
      Reverse genetic manipulation could be adopted as a com monly used technique. In 
      this study, we focus on using nonessential regions of NSP2 (aa480-532 and 
      aa508-532) as viral vector to express E2 Epitope of CSFV. A neutralizing epitope 
      of classical swine fever virus (CSFV) E2 protein was inserted into the two 
      nonessential region of nsp2 by the method of mutant PCR, basing on the infectious 
      clone of HuN4-F112 vaccine strain. The co-expressed full-length cDNA clones 
      (psk-HuN4-F112-delta508-532 + E2 and psk-HuN4-F112-delta480-532 + E2) were 
      assembled by cloning and splice of the gene fragments. The completely assembled 
      full-length cDNA clones were confirmed by sequence and Swa I enzyme digestion. 
      Capped RNAs were transcribed in vitro from a full-length cDNA clone of the viral 
      genome and transfected into BHK-21 cells by liposome to acquire the rescued 
      virus. The rescued recombinant viruses were passaged on MARC-145 cells. The 
      successfully rescued viruses were tested by RT-PCR, digestion, and genome 
      sequence. The results showed that these rescued viruses could be distinguished 
      from the parental virus (HuN4-F112) with the mutant genetic marker (Mlu I enzyme 
      site of virual genome at 14 667nt was created by synonymous mutation) and the 
      inserted nsp2 gene region. The results of IFA showed that the inserted E2 epitope 
      could be expressed by the recombinant viruses and the E2 epitope gene was stable 
      during the viral serial passage. The results of plaque assay and viral growth 
      curve showed that the recovery viruses possessed similar characterses of viral 
      growth to those of the parental virus. In summary, the full-length infectious 
      cDNA clones containing the marker gene were constructed and the marker 
      recombinant viruses were rescued. The results suggested that these stable 
      infectious clones could be used as an important tool for development of novel 
      vaccine against PRRSV.
FAU - Xu, Yan-Zhao
AU  - Xu YZ
AD  - Chinese Academy of Agricultural Sciences, Shanghai Veterinary Research Institute, 
      Shanghai 200241, China. haoxyz@163.com
FAU - Zhou, Yan-Jun
AU  - Zhou YJ
FAU - Tong, Wu
AU  - Tong W
FAU - Li, Ling
AU  - Li L
FAU - Jiang, Yi-Feng
AU  - Jiang YF
FAU - Tong, Guang-Zhi
AU  - Tong GZ
LA  - chi
PT  - English Abstract
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - China
TA  - Bing Du Xue Bao
JT  - Bing du xue bao = Chinese journal of virology
JID - 8803009
RN  - 0 (Epitopes)
RN  - 0 (Viral Envelope Proteins)
RN  - 0 (Viral Vaccines)
RN  - 0 (glycoprotein E2, classical swine fever virus)
RN  - EC 3.4.22.- (Cysteine Endopeptidases)
RN  - EC 3.4.22.- (nsP2 proteinase)
SB  - IM
MH  - Base Sequence
MH  - Cysteine Endopeptidases/*genetics
MH  - Epitopes/*genetics
MH  - Molecular Sequence Data
MH  - Porcine respiratory and reproductive syndrome virus/*genetics
MH  - Viral Envelope Proteins/genetics/*immunology
MH  - Viral Vaccines/immunology
EDAT- 2013/04/04 06:00
MHDA- 2013/06/14 06:00
CRDT- 2013/04/04 06:00
PHST- 2013/04/04 06:00 [entrez]
PHST- 2013/04/04 06:00 [pubmed]
PHST- 2013/06/14 06:00 [medline]
PST - ppublish
SO  - Bing Du Xue Bao. 2013 Jan;29(1):17-25.