蛋白激酶9在约氏疟原虫生长发育中的作用  

Study on the role of protein kinase 9 in the growth and development of Plasmodium yoelii

作　　者：程祥云 刘太平 陈穗林 徐文岳 CHENG Xiang-yun;LIU Tai-ping;CHEN Sui-lin;XU Wen-yue(Department of Pathogenic Biology,Army Medical University,Chongqing 400038,China)

机构地区：[1]陆军军医大学基础医学院病原生物学教研室

出　　处：《中国寄生虫学与寄生虫病杂志》2019年第3期248-253,共6页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家自然科学基金(No.81672053,No.2016XYY02)~~

摘　　要：目的探究蛋白激酶9 (PK9)在约氏疟原虫生长发育中的作用。方法基于疟原虫PlasmoDB数据库,查找约氏疟原虫致死株17XL (Py17XL)的PK9序列信息, BLAST比对Py PK9和恶性疟原虫PK9(Pf PK9)以及人蛋白激酶p78的同源性。提取Py17XL株野生型(WT)基因组DNA,设计引物, PCR扩增PK9基因。采用CRISPR-Cas9技术,将同源臂与sgRNA序列一起连接到PYC-MCS质粒中,构建PK9基因敲除(PK9KO)重组质粒。重组质粒电转至Py17XL虫株内,经乙胺嘧啶筛选、克隆,进行PCR鉴定和测序。10只雌性BALB/c小鼠随机分为WT感染组和PK9KO感染组,每组5只,重复3次实验。每鼠腹腔注射1×105个红内期疟原虫,每隔24 h取血检测小鼠原虫血症,观察小鼠存活情况。200只3~5日龄的斯氏按蚊随机分为WT感染小鼠吸血组和PK9KO感染小鼠吸血组,每组100只。于吸血8 d后解剖蚊胃,每组随机选取15只斯氏按蚊进行解剖,测定蚊胃中卵囊的数量。10只雌性BALB/c小鼠随机分为WT子孢子感染组和PK9KO子孢子感染组,每组5只,重复2次实验。每鼠尾静脉注射1×103个子孢子,每隔12 h取血,涂片观察其原虫血症出现时间。结果Py PK9与Pf PK9的序列一致性为82.5%, Py PK9和人类蛋白激酶p78的一致性为39.4%。PK9基因PCR扩增产物的长度约为597 bp。经PCR鉴定、测序确定PK9KO重组质粒构建成功, Py17XL虫株成功敲除PK9基因。WT感染组小鼠迅速出现原虫血症,感染后5~6 d全部死亡, PK9KO感染组小鼠全部存活,原虫血症在感染后17 d消失。WT感染小鼠吸血组和PK9KO感染小鼠吸血组的斯氏按蚊感染率分别为60.0%(9/15)和66.7%(10/15),差异无统计学意义(P> 0.05)。WT子孢子感染组和PK9KO子孢子感染组小鼠均于72 h查见原虫血症。结论PK9在红内期Py17XL的毒力中发挥了重要作用。Objective To explore the role of protein kinase 9(PK9) in the growth and development of Plasmodium yoelii. Methods The PK9 sequence information of P. yoelii lethal strain 17 XL(Py17 XL) was obtained from PlasmoDB. The sequence homology of PK9 among P. yoelii(Py PK9), P. falciparum(Pf PK9) and human protein kinase p78 was compared by BLAST. The primers were designed based on the Py PK9 DNA sequence and coding DNA was amplified from WT Py17 XL genomic DNA. The homologous arm and sgRNA sequence were ligated into PYC-MCS plasmid by CRISPR-Cas9 technique to construct PK9 gene knockout(PK9 KO) recombinant plasmid. The recombinant plasmid was transferred into Py17 XL by electroporation, screened and cloned by pyrimidine. The recombinant plasmid was identified by PCR and DNA sequencing. To determine the virulence of erythrocytic stage of PK9 KO P. yoelii, 5 female BALB/c mice were intraperitoneally infected with 1 × 105 PK9 KO P. yoelii erythrocytic stage. Another 5 mice were infected WT P. yoelii as control. Blood was taken every 24 h to detect parasitemia in mice, and the survival of the mice was observed( 3 repeated experiments). To determine the development of PK9 KO P. yoelii in mosquito, 200 Anopheles stephensi mosquitoes aged 3 to 5 days were randomly divided into two groups: WT group(n = 100) and PK9 KO group(n = 100), each was infected with WT P. yoelii or PK9 KO P.yoelii through feeding with infected mice. The mosquito stomach was dissected 8 days after being fed with blood,Each group was randomly selected from 15 Anopheles stephensi for necropsy, and the number of the developed oocysts in mosquito stomach was examined. To determine the infectivity of PK9 KO P. yoelii sporozoites developed in infected mosquitoes, 10 female BALB/c mice were randomly divided into WT sporozoite infection group( n = 5) and PK9 KO sporozoite infection group(n = 5). Each mouse was infected with 1 × 103 sporozoites through intravenous injection(2 repeated experiments). Tail vein blood was taken every 12 hours after injection, and th

关 键 词：约氏疟原虫 蛋白激酶9 红内期 

分 类 号：R382.319[医药卫生—医学寄生虫学]