猪瘟病毒云南毒株的分离鉴定及E2基因的原核表达

Isolation and Identification of Classical Swine Fever Virus in Yunnan Region and Expression of E2 Gene

从云南10个地州15个大型猪场采集到的21份样品中分离得到5株猪瘟病毒。经测序鉴定昆明、玉溪、曲靖地区分离株的核苷酸序列99.99%同源,大理和宝山地区的核苷酸序列99.99%同源,5株分离毒均属于基因二群。5株分离毒在PK-15细胞上的平均毒价约为2×106TCD50/mL,应用荧光抗体染色可以检测到CSFV。通过RT-PCR扩增猪瘟病毒约1 200 bp的E2蛋白全部抗原编码区序列,并将其分别克隆到pMD18-T载体中。序列分析结果表明,5株分离株的E2基因片段为1173bp,与猪瘟病毒Shimen株的核苷酸同源性为82.5%和84.3%,与C-株的核苷酸同源性分别为83.3%和85.4%。将E2基因插入原核表达载体pET-41a(+),转化进BL21(DE3)内进行表达,经IPTG诱导,E2蛋白能在菌体内高效表达,表达量达26.5%,蛋白分子质量约为41kD。本研究为猪瘟诊断抗原及基因工程疫苗的研制打下基础。

Five Classical swine fever virus strains were isolated from 21 samples in 11 prefectures of Yunnan region. The nucleo- tide homology of CSFV in KUNMING, YUXI and QUJING was 99.99%, and the sequence of CSFV in DALI shared a homology of 99.99% with BAOSHAN. All the 5 strains CSFV prevalent in YUNNAN were genotype II. The titers of the five strains virus were about 2 ×10^6TCID50/mL in PK - 15 cell. CSFV could be detected by using fluorescent antibody technique. The antigen coding full - length sequence of E2 genes about 1200 bp were obtained by RT - PCR, and which were cloned into pMD18 - T vectors. The results of sequence analysis showed that the E2 nucleotide fragments was 1 173 bp in length, The E2 sequence homology were 82.5% and 84. 3% in nueleotide acids compared with CSFV Shimen strain. The E2 gene was cloned into pET- 41a to construct the prokaryotic expression vector. The recombinant plasmid was transformed into BL21 （DE3） and expressed at high level induced by IPTG. The expressed E2 protein was amounted to 26.5% of the total bacteria proteins. The target protein was about 41 kD. This research laid a foundation for the research of immunological diagnosis and the genetic engineering vaccine for CSFV.