鸡补体C3d抗血清的制备与应用

Preparation and Application of Antiserum against Chicken Complement 3d

为探讨补体因子C3d在家禽健康诊断中的意义,将鸡补体因子C3d全长基因克隆到原核表达载体pET28a中构建重组表达质粒pET28a-C3d,然后将pET28a-C3d转化到大肠埃希菌BL21(DE3)并以IPTG诱导表达。表达产物采用氯化钾染色切胶法进行纯化,获得分子质量约35ku的C3d目的蛋白。将所得蛋白与弗氏佐剂制备疫苗免疫小鼠,制备抗鸡补体因子C3d血清。用制备的血清与重组杆状病毒表达的C3d蛋白进行Western blot和间接免疫荧光(IFA)鉴定血清的特异性。进一步应用该血清对不同健康状态的鸡血清进行C3d相关补体成份相对含量的检测。结果鸡补体因子C3d在大肠埃希菌中成功表达,纯化的C3d蛋白免疫小鼠后制备的抗血清经ELISA检测抗体效价达1∶256 000。Western blot和IFA结果显示,该血清能与重组杆状病毒表达的C3d蛋白发生特异性反应。临床应用试验表明,该血清不仅能用于检测不同健康状态鸡血浆中C3d的表达状况,还能用于ELISA检测不同鸡血清中C3d相关补体成分的相对含量差异。研究还证实鸡血浆中C3d相关补体成分的表达因机体免疫状态不同而存在差异,结果为了解C3d与鸡体健康状况之间的关系提供了依据。

To investigate the value of chicken complement 3d （C3d） in diagnosis of avian health, the full length of C3d gene was cloned into prokaryotic expressing plasmid pET28a and constructed recombinant expression plasmid pET28a-C3d. Then pET28a-C3d was transformed into BL21 （DE3） and induced by IPTG. The desirable C3d proteins with 35ku of molecular size were isolated by cutting the gel slices that contained the right band which was stained by KC1 solution. The vaccines were developed by mixing purified C3d proteins with Fred＇s adjuvant and inoculated into mice. The C3d antiserum raised from above vaccinated mice was identified by Western blot and immunofluoresence assay （IFA） based on C3d protein which was expressed in sf9 insect cells by recombinant baculovirus. The antiserum was further applied to detect C3d associated complement component relative levels of serum sampled from chickens in different health status. The results showed that the mouse serum against chicken C3d was developed by using C3d protein expressed in E. coli, which ELISA titre was above 1 ： 256 000. Western blot analysis and IFA showed that the antiserum could specifically bind with C3d protein expressed by recombinant baculovirus. The clinical application test showed that the antiserum, not only can be used in detection of C3d expression of chickens with different health status, can also be used in detection of C3d associated complement component relative levels of serum sampled from chicken in different health status by ELISA. Our results demonstrated that there were significantly different C3d associated complement component relative levels among chickens which were in different health status. It provided the evidence for learning the relationships between C3d associated complement component and chicken health status.