Preparation of the RNAse-resistant virus particles containing the partial gene fragments of avian influenza virus H5N1 and its application
10.3969/j.issn.1002-2694.2010.01.008
- VernacularTitle:含H5N1禽流感病毒核酸序列的病毒样颗粒的制备及应用
- Author:
Yuhua QI
;
Lunbiao CUI
;
Zhiyang SHI
;
Yiyue GE
;
Xian LI
;
Wenshuai ZHANG
;
Jun SHAN
;
Hua WANG
- Publication Type:Journal Article
- Keywords:
bacteriophage MS2;
H5N1 avian influenza virus;
virus-like particles;
prokaryotic expression vector
- From:
Chinese Journal of Zoonoses
2010;(1):29-32,35
- CountryChina
- Language:Chinese
-
Abstract:
To prepare the RNAse-resistant virus particles containing the partial gene fragments of avian influenza virus H5N1 for use as RNA standard and control in RNA virus detection, the genes coding the coat protein and maturase of E.coli bacteriophage MS2 were amplified by PCR and then cloned into prokaryotic expression vector pET32a to construct the intermediate vector pET32a-MS2. In addition, the gene sequences coding hemagglutinin (HA), neuraminidase(NA) and M protein of the H5N1 virus were also cloned separately to the down-stream of plasmid pET32a-MS2, thus constructing the prokaryotic expression vectors pET32a-NS2-HA, pET32a-MS2-NA and pET32a-MS2-M. These recombinant plasmids were then transformed separately to E.coli BL21(DE3) with induction by IPTG. to express the virus-like particles. The virus-like particles observed under electron microscopy were identified by RT-PCR ,while their stability was confirmed by real-time RT-PCR. In this way, the virus-like particles were successively constructed and identified through PCR amplification, enzymolysis identification and sequencing analysis. These virus-like particles observed under electron microscopy appeared to be circular in shape with a diameter of about 50 nm. Their stability was proved to be rather good. From these observations, it is apparent that these virus-like particles can be used as RNA standard and quality control in the detection of avian influenza virus H5N1.
