摘要
以自体免疫球蛋白作为外源抗原的载体来研制疫苗是一条研制安全、高效疫苗的新途径。将猪IgGH链基因中的CDR3区缺失 ,剩余的两个片段分别连接到pBluescriptⅡSK(+)和 pBluescriptⅡKS(+)上 ,其中一片段再与编码FMDVVP1两个拷贝的 141～ 16 0位氨基酸残基和一个拷贝的 2 0 0～ 2 13位氨基酸残基构成的串联片段2 0AA～ 14AA～ 2 0AA的核苷酸片段相连。然后将它们切下与质粒表达载体 pET2 8b相连 ,构建出融合表达质粒pET2 8b FH ,经限制酶酶解及DNA序列分析证明该融合基因各连接点及读框正确。转入大肠杆菌BL2 1(DE3) plysS表达出一分子量为 6 4kD的蛋白质。经ELISA检验证明 ,大肠杆菌细胞中表达的融合蛋白有O型FMDV抗原活性。将该融合蛋白肌肉注射免疫豚鼠和猪 ,攻毒实验表明 ,疫苗pET2 8b FH对豚鼠产生了保护作用 ,保护率为 6 6 % ,对猪也有保护作用。
It is a new approach to use self immunoglobulin molecule as carriers for the foreign epitopes to prepare for effective and safe vaccines. Recombinant vaccine study on foot\|and\|mouth disease virus proved that the fusion protein containing a β\|galactosidase from E.coli and the two copies of amino acid residue 141～160 and one copy of amino acid residue 200～213 of FMDV type O elicited high levels of neutralizing antibody and protected both guinea pigs and swine against viral challenge. In this study using swine self IgG H chain as a carrier protein to construct a recombinant vaccine against FMDV type O. Firstly,the CDR3 region of the V\|region of swine IgG H chain was deleted by PCR techniques. Secondly,the partial V\|region and FR 4 C\|region were cloned into pBluescript Ⅱ SK(+) and pBluescript Ⅱ KS(+) respectively. Thirdly, a tandem repeat nucleotide fragment encoded 20AA～14AA～20AA of VP1 of FMDV O Type was ligated at the 3′ end of partial V\|region. Finally,partial V\|region 20AA～14AA～20AA and FR 4\|C\|region were cut and isolated respectively, and ligated into prokyryotic expression vector p ET 28b . Desired recombinants were obtained by restriction endonucleases and partial sequence analysis and termed p ET 28b FH. A 64kD protein was expressed in E.coli. Western blot and ELISA assay showed that the immunogenicity of expressed fusion protein was positive. Guinea pigs were intramuscularly inoculated with 200μg of p ET 28b FH fusion protein at two weeks intervals for two times. 66% of guinea pigs were protected against FMDV Type O infection.Swines were intramuscularly inoculated with 1.5mg of p ET 28b FH fusion protein at two weeks intervals for two times,and the symptom had been postponed for six days compared with the control.
出处
《生物工程学报》
CAS
CSCD
北大核心
2000年第6期679-683,共5页
Chinese Journal of Biotechnology
基金
科技部生物工程中心中试开发处下达的 863课题!( 19972 10 )
关键词
基因工程疫苗
O型口蹄疫病毒
构建
FMDV,genetic engineering vaccine,self immunoglobulin,swine IgG H chain
