大劣按蚊含硫脂蛋白基因在约氏疟原虫感染中的作用

Function of TEP1 Gene during Plasmodium yoelii Infection in Anopheles dirus

目的分析大劣按蚊含硫脂蛋白(TEP1)基因在约氏疟原虫感染过程中的作用。方法根据GenBank中大劣按蚊TEP1基因序列设计带有T7启动子的引物,以大劣按蚊cDNA为模板,进行PCR扩增,纯化产物,体外转录试剂盒合成AdTEP1双链RNA。羽化1～2日的雌性大劣按蚊分3组(200只/组):TEP1干扰组、绿色荧光蛋白(EGFP)干扰组和对照组。TEP1、EGFP干扰组按蚊分别进行胸部微量注射AdTEP1双链RNA、EGFP双链RNA各147 ng,对照组未处理。干扰后3d,每组取15只按蚊,去头,以大劣按蚊核糖蛋白S7(AdS7)为内参进行半定量PCR,检测干扰效果。干扰后4d,用约氏疟原虫BY256荧光株感染3组大劣按蚊。感染后9d,每组各解剖25只蚊胃,记录感染率和感染度。结果对照组和EGFP干扰组AdTEP1的表达条带亮度基本一致,而TEP1干扰组AdTEP1的表达条带亮度非常微弱。感染后9 d,蚊胃卵囊数统计学分析表明,对照组、EGFP干扰组和TEP1干扰组感染率分别为(24±2.83)%、(24±0.71)%和(80±3.54)%;感染度分别为0.32±0.7、0.44±0.85和5.52±4.84,TEP1干扰组明显高于对照组和EGFP干扰组(P<0.05)。结论 AdTEP1干扰后明显增加了大劣按蚊的感染率和感染度,增强了大劣按蚊对约氏疟原虫易感性。

Objective To study the role of TEP1 gene from Anopheles dirus during Plasmodium yoelii infection by RNA interference.Methods TEP1 primers with T7 promoter were designed based on the sequence of An.dirus TEP1 gene from GenBank database.PCR amplification of TEP1 gene was completed with An.dirus cDNA as template.The AdTEP1 double-stranded RNA was synthesized by using in vitro transcription kit with purified PCR products.Female An.dirus emerged for 1-2 days were divided into three groups each with 200 mosquitoes： TEP1 interference group,EGFP interference group and control.Mosquitoes in TEP1 and EGFP interference groups were microinjected in chest with 147 ng of AdTEP1 and EGFP double-stranded RNA,respectively,while those of control group were untreated.Effect of TEP1 interference on P.yoelii in An.dirus was estimated through semi-quantitative PCR with internal reference AdS7 at 3 d after injection.On 4 d after injection,mosquitoes were infected by EGFP-expressing P.yoelii BY265.The infection rate and infectiosity of mosquitoes were observed through anatomizing 25 midguts from each group at 9 d post-infection.Results The AdTEP1 double-stranded RNA did well in the interference of TEP1 expression in An.dirus.The infection rate in the groups of control,EGFP and TEP1 interference was（24±2.83）%,（24±0.71）%,and（80±3.54）%,respectively;and the infectiosity of the three groups was 0.32±0.7,0.44 ± 0.85,and 5.52 ± 4.84,respectively.Conclusion AdTEP1 interference increases the infection rate and infectiosity of An.dirus by P.yoelii,and raises the susceptibility of An.dirus to P.yoelii significantly.TEP1 plays a critical role in the process of P.yoelii infection.