高致病性猪繁殖与呼吸综合征病毒非结构蛋白Nsp4的表达及其抗血清的制备

根据GenBank中已发表的高致病性猪繁殖与呼吸综合征病毒（Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus，PRRSV）HuN4株全基因组序列，设计合成一对引物，对PRRSV HuN4株非结构蛋白Nsp4（Nonstructural protein 4基因进行RT-PCR扩增，克隆到原核表达载体pET30a（＋）中，构建重组表达载体pET30a-Nsp4，经酶切测序鉴定。将pET30a-Nsp4转化表达菌株BL21（DE3），诱导可表达分子量约为27kDa重组蛋白。Westernblot显示其具有较好的反应原性，经镍离子亲和层析（Ni-NTA）纯化获得了高纯度的可溶性重组蛋白。将纯化的Nsp4蛋白免疫BALB／c小鼠，抗血清ELISA效价达1：16000，Western blot和IFA表明，所制备的抗血清能够特异性识别PRRSV自身表达的Nsp4蛋白。本研究获得了可溶性的PRRSV Nsp4蛋白，制备了Nsp4特异性多抗血清，为进一步研究Nsp4蛋白的亚细胞定位及功能奠定了基础。

PROKARYOTIC EXPRESSION OF NONSTRUCTURAL PROTEIN 4 (NSP4) OF HIGHLY PATHOGENIC PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS AND CHARACTERIZATION OFANTI-NSP4 POLYCLONAL ANTIBODY

The nonstructural protein 4 （Nsp4） gene of highly pathogenic porcine reproductive and respiratory syndrome virus （HP-PRRSV） HuN4 strain was amplified by RT-PCR with a pair of specific primers designed from the sequence deposited in GenBank, and then the product was cloned into pET30a （ ＋ ） vector and sequenced. The resulting plasmid pET30a Nsp4 was transformed into Escherichia coli BL21 （DE3） for expression with induction of IPTG. The SDS-PAGE analysis revealed that the molecular weight of the expressed product was 27 kDa. The expressed protein was purified using Ni-NTA affinity chromatography. Western blot analysis showed that the purified protein was specifically recognized by swine antiserum against the HP-PRRSV. The antiserum specific for Nsp4 were generated by immunizing BALB/c mice using the purified protein. The titer of the murine antiserum was about 1 ： 16 000 as detected by ELISA. The indirect immunoflourescence assay and Western blot demonstrated that the murine antiserum reacted with the native HP-PRRSV Nsp4.