伯氏疟原虫推测分泌动合子蛋白7截短片段传播阻断功能的研究

doi:10.12140/j.issn.1000-7423.2019.05.003

中国寄生虫学与寄生虫病杂志 ›› 2019, Vol. 37 ›› Issue (5): 520-525.doi: 10.12140/j.issn.1000-7423.2019.05.003

伯氏疟原虫推测分泌动合子蛋白7截短片段传播阻断功能的研究

郑文琪^1,²(), 王俊瑞¹, 王华², 刘飞³, 罗恩杰⁴, 曹雅明³, 韩艳秋^1,^*()

1 内蒙古医科大学附属医院临床检验中心,呼和浩特 010050
2 内蒙古医科大学基础医学院寄生虫学教研室,呼和浩特 010050
3 中国医科大学基础医学院,免疫学教研室,沈阳 110021
4 中国医科大学基础医学院,病原生物学教研室,沈阳 110021

收稿日期:2019-03-11 出版日期:2019-10-30 发布日期:2019-11-07
通讯作者: 韩艳秋
作者简介:
作者简介：郑文琪（1988-）,女,博士,助理研究员。从事疟原虫的抗感染免疫方面研究。E-mail：zhengwenqi2011@163.com
基金资助:
国家自然科学基金（No. 81760367）;内蒙古自治区卫生和计划生育委员会科研计划项目（No. 201703106）;内蒙古医科大学附属医院博士启动基金（No. NYFYBS2018）

Truncated Plasmodium berghei putative secreted ookinete protein 7 induces transmission-blocking immunity

Wen-qi ZHENG^1,²(), Jun-rui WANG¹, Hua WANG², Fei LIU³, En-jie LUO⁴, Ya-ming CAO³, Yan-qiu HAN^1,^*()

1 Department of Clinical Laboratory, Affiliated Hospital of Inner Mongolia Medical University, Hohhot 010050, China
2 Department of Parasitology, Basic Medicine College, Affiliated Hospital of Inner Mongolia Medical University, Hohhot 010050, China
3 Department of Immunology, College of Basic Medical Sciences, China Medical University, Shenyang 110021, China
4 Department of Pathogen Biology, College of Basic Medical Sciences, China Medical University, Shenyang 110021, China

Received:2019-03-11 Online:2019-10-30 Published:2019-11-07
Contact: Yan-qiu HAN
Supported by:
Supported by the National Natural Science Foundation of China (No. 81760367）, the Scientific Research Project of Health and Family Planning Commission of Inner Mongolia (No. 201703106）, and the Doctoral Scientific Research Project of Affiliated Hospital of Inner Mongolia Medical University (No. NYFYBS2018）

摘要/Abstract

摘要：

目的通过体外阻断实验与蚊虫直接喂养实验对截短的重组伯氏疟原虫推测分泌动合子蛋白7（rPbPSOP7）31~210氨基酸（aa）片段的传播阻断能力进行研究。方法体外阻断实验,雌性BALB/c小鼠经腹腔注射5 × 10⁶伯氏疟原虫感染红细胞（PbRBC）,感染后第3天,尾静脉收集血液,与rPbPSOP7免疫小鼠血清（大肠埃希菌表达并纯化,与弗氏佐剂混合后免疫BALB/c小鼠）进行混合培养,观察不同稀释倍数（1 : 5、1 : 10、1 : 50）免疫血清培养15 min后疟原虫配子体出丝数及培养24 h后动合子形成数的变化,以同样稀释倍数的His标签蛋白免疫血清为对照。蚊虫直接喂养实验,rPbPSOP7（实验组）或His标签蛋白（对照组）与弗氏佐剂混合后各免疫5只雌性BALB/c小鼠,于末次免疫后第10天,用苯肼处理并经腹腔注射5 × 10⁶ PbRBC,感染后第3天,每组小鼠用至少50只雌性按蚊直接吸食小鼠血液,吸血后第10天解剖按蚊,计数蚊胃壁上的囊合子数,计算蚊虫感染率。实验重复3次。结果体外阻断实验中,与His标签蛋白免疫血清对照组相比,rPbPSOP7免疫血清1 : 5、1 : 10和1 : 50稀释培养的配子体出丝数分别减少了22.0%、8.0%和2.0%,差异无统计学意义（P > 0.05）;与对照组相比,培养24 h后,1 : 5、1 : 10与1 : 50稀释培养的动合子形成数分别减少了69.2%、56.4%和48.6%（P < 0.01）,呈抗体剂量依赖关系。3次独立的蚊虫直接喂养实验结果显示,实验组每只蚊胃壁上的平均囊合子数分别为（31.1 ± 34.9）、（30.2 ± 32.3）和（29.1 ± 35.1）个,较对照组的（87.1 ± 69.3）、（96.4 ± 82.9）和（85.3 ± 69.1）个分别减少了64.2%、68.6%和65.8%（P < 0.01）。按蚊感染率由对照组的96.4%（27/28）、89.3%（25/28）和89.3%（25/28）,降至实验组的78.6%（22/28）、78.6%（22/28）和75%（21/28）,分别降低了17.8%、10.7%与14.3%（P > 0.05）。结论 rPbPSOP7免疫血清可明显减少动合子及囊合子的形成,抗rPbPSOP7血清具有一定的传播阻断能力。

关键词: 伯氏疟原虫, 传播阻断疫苗, 候选抗原, 推测分泌动合子蛋白7

Abstract:

Objective To investigate the truncated Plasmodium berghei putative secreted ookinete protein 7 (PbPSOP7) induces transmission-blocking immunity determined by in vitro antibody blocking and mosquito feeding assays. Methods Recombinant PbPSOP7 protein (rPbPSOP7) was expressed in E. coli and the anti-rPbPSOP7 sera were obtained by immunizing BALB/c mice for three times with rPbPSOP7 formulated with Freund’s adjuvant. For in vitro test, P. berghei-infected mouse blood was obtained from a mouse infected with 5 × 10⁶ P. berghei for three days, then co-incubated with mouse anti-rPbPSOP7 sera at 1 : 5, 1 : 10 and 1 : 50 dilutions. Mouse anti- a non-relevant His-tag protein serum at the same dilutions was used as control. The inhibition on the exflagellation of Plasmodium gametocytes was observed 15 min after the culture and the inhibition on the ookinete development was observed 24 hours after incubation. For mosquito feeding experiment, 5 mice were each immunized with 50 μg of rPbPSOP7 formulated with Freund’s adjuvant for three times, then infected with 5 × 10⁶ P. berghei by intraperitoneal injection 10 days after the last immunization. Three days post-infection, the mice were fed by at least 50 female anopheles mosquitoes. The mosquitoes were dissected 10 days after the blood feeding, the infection rate and the reduced number of oocysts in each infected mosquito were calculated compared with mosquitoes fed with mice immunized with His-tag control protein. The feeding experiment was repeated three times. Results When incubated with mouse anti-rPbPSOP7 sera at the 1 : 5, 1 : 10 or 1 : 50 dilutions for 15 min, the exflagellation of gametocytes was decreased by 22.0%, 8.0% and 2.0% compared to Plasmodium incubated with sera from mice immunized with His-tag protein(P > 0.05). After incubated with mouse anti-rPbPSOP7 sera for 24 hours, the significant inhibition of ookinete development was observed in the culture with different dilutions of the antisera. The development of ookinete was reduced by 69.2%, 56.4% and 48.6% when incubated with anti-rPbPSOP7 sera at 1 : 5, 1 : 10 and 1 : 50 dilutions, respectively, compared with control sera group (P < 0.01). Three independent mosquito feeding experiments showed that, compared with the control group, the development of oocysts in the midgut of mosquitoes in immunized group was significantly decreased from 87.1 ± 69.3, 96.4 ± 82.9 and 85.3 ± 69.1 to 31.1 ± 34.9, 30.2 ± 32.3 and 29.1 ± 35.1, respectively. Anti-rPbPSOP7 sera reduced the development of oocyst by 64.2%, 68.6% and 65.8% in three independent feeding experiments (P < 0.01). The infection rates of P. berghei in mosquitoes decreased from 96.4% (27/28), 89.3% (25/28) and 89.3% (25/28) to 78.6% (22/28), 78.6% (22/28) and 75.0% (21/28) in three experiments, with 17.8%, 10.7% and 14.3% reduction (P > 0.05). Conclusion Immunization of rPbPSOP7 induced transmission-blocking immunity characterized by reduced development of P. berghei ookinate and oocyst in mosquitoes, indicating that PbPSOP7 is a potential malaria transmission-blocking vaccine candidate.

Key words: Plasmodium berghei, Transmission-blocking vaccines, Candidate antigen, Putative secreted ookinete protein 7

中图分类号:

R531.3

郑文琪, 王俊瑞, 王华, 刘飞, 罗恩杰, 曹雅明, 韩艳秋. 伯氏疟原虫推测分泌动合子蛋白7截短片段传播阻断功能的研究[J]. 中国寄生虫学与寄生虫病杂志, 2019, 37(5): 520-525.

Wen-qi ZHENG, Jun-rui WANG, Hua WANG, Fei LIU, En-jie LUO, Ya-ming CAO, Yan-qiu HAN. Truncated Plasmodium berghei putative secreted ookinete protein 7 induces transmission-blocking immunity[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2019, 37(5): 520-525.

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参考文献 19

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伯氏疟原虫推测分泌动合子蛋白7截短片段传播阻断功能的研究

Truncated Plasmodium berghei putative secreted ookinete protein 7 induces transmission-blocking immunity

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