摘要
以pVAX1为载体首次构建了犬冠状病毒病基因的3种真核表达质粒。首先将犬冠状病毒大熊猫株(CCV DXMV)纤突蛋白(S)、膜蛋白(M)和核蛋白(N)基因进行了克隆和序列测定。将测序后的质粒pTS、pTM和pTN分别双酶切,回收目的基因片段并将其定向克隆到真核表达质粒pVAX1中得到重组质粒pVAXS、pVAXM和pVAXN。将这3种真核表达质粒通过脂质体介导法转染MDCK细胞,通过RT-PCR法进行转录水平的检测,并用间接ELISA法检测目的蛋白的表达情况。结果在pVAXS、pVAXM、pVAXN转染MDCK细胞36 h后就可检测到目的基因的转录;在转染72 h后可检测到3种目的蛋白的表达。动物免疫试验表明,3种真核表达载体能有效地诱导机体产生细胞免疫与体液免疫应答,这为CCV基因疫苗的研究奠定了良好的基础。
In this experiment, three recombinant expression vectors based on pVAX1 were successfully constructed. According to the sequences of spike （S） gene, membrane protein （M） gene and nucleoprotein （N） gene of CCV Insavc-1 strain, three pairs of specific primers were designed and used to amplify S, M and N genes of DXMV strain. The PCR products were cloned into pGEM-T. Then the recombinant pTS, pTM and pTN were used for sequencing. After sequenced, three immunogenicial genes were subcloned into pVAX1, respectively, which named pVAXS, pVAXM and pVAXN. The three constructed expression vectors were transfected into MDCK cell. Transcriptions of interesting genes were detected by RT-PCR. The expression of target proteins was detected by indirect ELISA. The results showed that target genes can be transcripted after 36 hours , and target proteins can be detected after 72 hours of transformation.Immunization in dogs indicated that the three expression vectors can efficiently induce the specific cellular and humoral immunoresponse, which laid a solid foundation for the further studies of new vaccines of CCV.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2006年第4期412-416,共5页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
国家自然科学基金资助项目(30000123)
