柔嫩艾美耳球虫热休克蛋白70(Et HSP70)的重组表达及免疫保护效果

本研究初步评价了柔嫩艾美耳球虫热休克蛋白70(EtHSP70)的免疫保护效果。以RT-PCR方法克隆获得E.tenella广东株的Ethsp70基因序列,插入表达载体pMAL-c2X后转化入大肠杆菌Rosetta株,经IPTG诱导,可高效表达分子量为112 ku的可溶性融合蛋白,表达量约占菌体总蛋白的27%。将Ethsp70基因插入真核载体pcDNA6,构建真核表达质粒pcDNA-Ethsp70。用纯化的重组融合蛋白(rEtHSP70)和pcDNA6-Ethsp70质粒分别以100μg/只剂量肌注免疫雏鸡,攻虫后以盲肠病变计分、相对盲肠卵囊产量(ROP)、相对增重率和抗球虫指数(ACI)为评价指标,结果表明2个免疫组相对盲肠卵囊产量分别为39.4%、45.2%,抗球虫指数由89分别提高至免疫组的164和150。提示EtHSP70可能是一种有潜在应用价值的保护性抗原。

Recombinant expression of heat shock protein 70 of Eimeria tenella and its protective immunity

The immuno-protective effects of heat shock protein 70 from Eimeria tenella(EtHSP70) was evaluated in this study.The EtHSP70 gene fragment was obtained by RT-PCR with a pair of specific primers designed according to Ethsp70 gene sequence in GenBank,and inserted into the plasmid pMAL-c2X and pcDNA6 for constructing recombinant expressing vector pMAL-c2X-Ethsp70 and pcDNA6-Ethsp70,respectively.Recombinant pMAL-c2X-Ethsp70 was induced with IPTG to express the fusion EtHSP70 in the transformed E.coli Rosseta strain.Recombinant EtHSP70 with a molecular weight of 112 ku accounted for 27% of the total bacterial proteins.Every chicken in different groups was separately vaccinated with 100 μg of recombinant fusion EtHSP70 or pcDNA6-Ethsp70 at 14 and 21 day-old,and challenged with sporolated occysts of E.tenella at 28 day-old.Some indexes,including relative weight gain,relative oocyst production(ROP) in caeca,and anticoccidial index(ACI),were used to evaluate the immune effect.The results showed that the ROP is 39.4% and 45.2% in rEtHSP70 vaccinated group and pcDNA6-Ethsp70 immunized group,respectively.It suggested that partial protection against the challenge of E.tenalla oocysts could be triggered after the chicken was vaccinated with the vaccines based on EtHSP70.