鸡CTLA-4胞外区的原核表达与免疫佐剂作用

为了探索鸡细胞毒性T细胞相关抗原-4(CTLA-4)胞外区(ChIgV)作为免疫佐剂的可行性,采用RT-PCR从鸡外周血淋巴细胞扩增ChIgV编码序列;测序结果显示,其核苷酸序列与已发表序列的同源性为100%。将猪CD151基因片段克隆入原核表达载体pET-30a和ChIgV融合表达载体pET-ChIgV,获得重组表达载体pET-CD151和pET-ChIgV-CD151。分别将pET-CD151和pET-ChIgV-CD151转化BL21(DE3)大肠杆菌,用IPTG诱导重组菌表达,SDS-PAGE结果显示,能表达预期的18ku和28ku重组蛋白,表达产物均为不溶性包涵体。采用包涵体纯化法和尿素变性/复性法获得了纯化的可溶性重组蛋白。以每只500μg剂量2次免疫SPF鸡,用间接ELISA测定血清抗体效价,结果显示,CD151免疫组在初免后第3周特异抗体检测为阳性,第5周抗体效价达到1∶13 000;ChIgV-CD151免疫组在初免后第2周特异抗体检测为阳性,第4周抗体效价达1∶53 000。分别用CD151和ChIgV-CD151免疫血清进行Western-blot检测,结果显示,均能在CD151阳性猪肺巨噬细胞膜蛋白中检测到预期的29ku蛋白条带,而在CD151阴性PK-15细胞膜蛋白中不能检测到相应的蛋白条带。表明,ChIgV可作为鸡抗原提呈细胞的靶向导入分子和新型免疫佐剂。

Prokaryotic expression and immunoadjuvant effect of the extracellular domain of chicken CTLA-4

To investigate the adjuvant effect of the extracellular domain(ChIgV) of chicken cytotoxic T lymphocyte-associated antigen 4,ChIgV-coding sequence was amplified from peripheral blood lymphocytes using RT-PCR and subcloned into prokaryotic expression vector pET-30a,resulting in ChIgV fusion expression vector pET-ChIgV.The cDNA fragment of porcine CD151 was inserted into pET-30a and pET-ChIgV,respectively,and the resultant recombinant vector pET-CD151 or pET-ChIgV-CD151 was transformed into BL21(DE3) E.coli.After being inducted with IPTG,the expected 18 ku or 28 ku protein band was detected in pET-CD151 or pET-ChIgV-CD151 transformant by SDS-PAGE,which was present mainly in the form of inclusion bodies.After repeatedly washing and denaturation/renaturation in urea-containing buffer,the purified soluble recombinant proteins were obtained.Each SPF chicken was immunized two times with 500 μg recombinant CD151 or ChIgV-CD151 fusion protein with the serum antibody was titrated using ELISA.For CD151-immunized group,the specific antibody was detectable from week 3 and reached to the maximal titer of 1∶13 000 on week 5 after primary immunization.For ChIgV-CD151-immunized group,however,the specific antibody was detectable from week 2 and reached the maximal titer of 1∶53 000 at week 4 after primary immunization.Western-blot showed that the two anti-sera could be detected the expected 29ku protein band in CD151-positive pig alveolar macrophages,but not in CD151-negative PK-15 cells.The results suggested that ChIgV can target its fusion protein to chicken antigen presenting cells and thus can be used as a new type of immune adjuvant.