猪带绦虫TSOL18-SP蛋白的原核表达及其多抗的制备

对TSOL18基因中4个碱基进行定点突变,并截去N端16个氨基酸的信号肽,构建pGEX-TSOL18-SP原核表达载体,IPTG诱导表达后进行产物的SDS-PAGE及Western blot分析;用改造的纯化蛋白免疫家兔后采集血清,经琼脂双扩散试验检测血清抗体效价.结果表明:TSOL18基因改造后长度为345 bp,共有7个碱基发生突变,但氨基酸序列没有改变,与GenBank收录的TSOL18基因核苷酸序列相似性为97%,氨基酸序列相似性为100%.SDS-PAGE及Western blot分析表明:改造的重组菌诱导后可高效表达可溶性目的蛋白,且TSOL18-SP分子量约为38.7 kD,与猪带绦虫六钩蚴阳性血清反应性强;琼脂双扩散法测定免疫兔血清抗体效价为1∶32~64,说明表达的TSOL18-SP蛋白具有较好的免疫原性.

Procaryotic expression and polyclonal antibody preparation of Taenia solium TSOL18-SP protein

In order to improve the biologic activity and stability of the recombinant 18 kD antigen(TSOL18) of Taenia solium onchospere,the expressive vector of pGEX-TSOL18-SP was constructed with the modified T.solium onchospere TSOL18 gene by the rite-directed mutagenesis of 4 bases in its coding sequence and removing of the sequence fragment encoding its N-terminal signal peptide.After induced with IPTG,the expressed proteins were analyzed by SDS-PAGE and Western-blot.The rabbits were immunized with the purified TSOL18-SP protein and the titer of serum antibody was detected by agar double diffusion test.It was shown that seven bases were mutated in the reconstructed TSOL18 gene with 345 bp open reading frame.Compared with the published sequences of 18 kD protein of T.solium oncosphere(GenBank accession number AF017788),its relative similarities in nucleotide and amino acids were 97 % and 100 %,respectively.Molecular mass of TSOL18-SP was approximately 38.7 kD on SDS-PAGE and reacted well with mice positive serum against T.solium onchospere.The antibody titers of rabbit against TSOL18-SP reached up to 1∶32～64 by agar double diffusion test.The results indicated that the TSOL18-SP protein has a better immunogenicity and it could be used for the vaccine development of cysticercosis.