罗非鱼补体C3基因的克隆及其表达分析

Cloning and tissues expression analysis of complement C3 of tilapia

[目的]获得罗非鱼补体C3并了解其在罗非鱼免疫前后各组织中的表达特征。[方法]通过PCR扩增获得补体C3片段,通过DNA Star、MEGA 6.06等分析其序列及理化特性;将经纯化获得的Scp B蛋白以5μg/g的剂量免疫罗非鱼,利用实时荧光定量PCR测定C3在各组鱼体的表达变化。[结果]补体C3基因c DNA序列6 994 bp,ORF全长5 361 bp,共编码1 786个氨基酸。其在罗非鱼肝脏中表达量最高,且免疫组肝脏中C3表达量在感染后4h时显著高于对照组（p〈0.05）;此外,感染前（0 h）免疫组头肾和脾脏中C3表达量均显著低于对照组（p〈0.05）,但在感染后4~12h,免疫组表达量均呈升高趋势,而对照组则呈降低趋势。[结论]抗原免疫可提高罗非鱼感染无乳链球菌后组织中补体C3的表达量,补体C3在罗非鱼抵御无乳链球菌感染过程中发挥重要作用。

[ Objective] To clone the complement C3 gene of tilapia and analyze the expression profiles in vaccinated and non - vaccinated tilapia. [ Methods ] The cDNA sequence of complement C3 was amplified by polymerase chain reaction （PCR）. Its physicochemical properties were analyzed through DNA Star and MEGA 6.06. Tilapias were vaccinated with protein ScpB with the dose of 5 Ixg/g and the expression profile of complement C3 in vaccinated and non - vaccinated tilapia was tested u- sing real time PCR. [Results]The 6 994bp of the complement C3 cDNA sequence was obtained and the open reading frame （ORF） was 5 361bp which encoded 1 786 amino acids. The highest expression of complement C3 was detected in liver. At the first 4 hours, the expression quantity of the complement C3 in the liver of immunity group was significantly higher than the control group （p 〈0.05）. The expression quantity of the complement C3 in head -kidney and spleen was significantly lower than the control group before infection （p 〈0.05）. However,at 4 - 12 h after infection,the expression quantity of the complement C3 in the immunity group showed increasing trend and that of in the control group showed decreasing trend. Meanwhile, the ex- pression quantity of the complement C3 in spleen was significantly higher than that of in the control group at the 4 h after infec- tion （p 〈 0.05 ）. [ Conclusion] The active immunization could increase the expression of complement C3 in tilapia and the complement C3 played an important role in mediating the response protection against S. agalactiae in Nile tilapia.