刚地弓形虫肌动蛋白基因的克

发布时间：2018-06-09 03:39

本文选题：刚地弓形虫 + 肌动蛋白　；参考：《山西医科大学》2012年硕士论文

【摘要】：实验目的对刚地弓形虫肌动蛋白(TgACT)基因进行克隆、表达、纯化,分析其抗原性,观察不同剂量rTgACT免疫小鼠诱导的黏膜及系统免疫应答及最佳剂量免疫小鼠的抗弓形虫感染的能力,探讨TgACT作为弓形虫疫苗候选抗原的可能性。实验方法第一部分：构建重组质粒pET30a-TgACT,并在大肠杆菌中高效表达。收集、纯化RH株弓形虫速殖子,提取总RNA;设计合成引物并引入EcoRI和Xho Ⅰ酶切位点,RT-PCR扩增编码TgActin的基因片段克隆到原核质粒pET30a中,经双酶切、PCR及测序鉴定阳性克隆；在大肠杆菌BL21/DE3中用IPTG诱导表达,表达产物经SDS-PAGE进行鉴定,通过稀释复性的方法获得大量表达蛋白,以Ni-NTA层析法纯化蛋白,用兔抗弓形虫血清做Westen blotting分析其抗原性。第二部分：观察不同剂量rTgACT滴鼻免疫小鼠诱导的黏膜和系统免疫应答。BALB/c小鼠50只随机分为5组,免疫组分别用10μg、20μg、30μg、40μgrTgACT/只滴鼻免疫小鼠3次,首免与二免间隔2周,二免后1周三免,rTgACT溶于20μl PBS中,对照组用等量PBS滴鼻。三免后第14d,眼静脉丛采血,分离血清。颈椎脱臼处死小鼠,采集小肠冲洗液、鼻咽和阴道冲洗液。ELISA法测定上述3种冲洗液IgA和血清IgG。分离脾淋巴细胞计数并培养,ELISA法测定脾细胞培养液上清中IL-2和IL-4(24h)及IFN-γ(96h)水平。用rTgACT不(?)ConA刺激脾淋巴细胞,CCK-8试剂盒测定其增殖程度。确定rTgACT的最佳免疫剂量。第三部分：观察30μ rTgACT滴鼻免疫小鼠诱导的抗弓形虫感染保护作用。BALB/c小鼠40只随机分为2组,用30μg rTgACT溶于20μl PBS中滴鼻免疫小鼠3次,首免与二免间隔2周,二免后1周三免,,对照组用等量PBS滴鼻。末次免疫后第14d,用8×104个速殖子/只灌胃攻击全部小鼠,观察小鼠健康状况。攻击后第30d,颈椎脱臼处死小鼠,分离计数肝、脑组织内弓形虫速殖子。实验结果第一部分：从弓形虫RH株cDNA中扩增出1131bp的TgACT基因片段,并成功构建重组质粒pET30a-TgACT;SDS-PAGE结果表明,目的基因在大肠杆菌BL21/DE3中以包涵体形式高效表达,相对分子量(Mr)约49kDa,比预期值大约7kDa。通过稀释复性的方法获得大量表达蛋白,经Ni-NTA纯化后的蛋白浓度较低。用超滤浓缩管浓缩蛋白以提高蛋白浓度。Western blotting显示纯化后的rTgACT能被兔抗弓形虫免疫血清识别。第二部分：不同剂量rTgACT滴鼻免疫小鼠,与对照组相比,均不同程度诱导了小鼠特异性免疫应答。血清弓形虫特异性IgG,30μg、40μg组与对照组差异显著,鼻咽、阴道冲洗液特异性IgA30μg组高与对照组(P0.05)。小鼠脾淋巴细胞增殖实验结果各组之间也无显著性差异。IL-2各组之间变化无显著差异；IL-4,与对照组相比,10gg、20μg、30μg、40μg组均增高(P0.05),各免疫组之间无显著差异；IFN-γ,与对照组相比,10μg、20μg、30μg、40μg组均增高(P0.05),各免疫之间无显著差异。总之,20μg、30μg、40μg都可以诱导免疫应答反应,30μg组可以更有效地诱导粘膜和系统免疫应答。综合分析,确定30μg为较理想免疫剂量。第三部分：30μg rTgACT与PBS滴鼻免疫小鼠,末次免疫2周后,用8×104个速殖子/只灌胃攻击全部小鼠攻击后第2-20d,小鼠出现竖毛、倦怠、活动减少、饮水及采食减少等异常表现。对照组小鼠死亡10只,免疫组死亡10只。免疫组肝组织内虫荷(速殖子数)77.89±20.00×105/g显著低于对照组肝组织内虫荷(速殖子数)169.77±59.74×105/g(P0.05),减虫率为54.12%；脑组织内虫荷(速殖子数)13.20±2.54×105/g,显著低于对照组22.29±3.35×105/g(P0.05),减虫率为40.78%。30μg组rTgACT滴鼻免疫可以有效地抗弓形虫感染。结论成功构建重组质粒pET30a-TgACT并在大肠杆菌中高效表达,rTgACT具有抗原性。30μg组rTgACT滴鼻免疫更有效诱导了黏膜和系统免疫应答,并有效抵抗弓形虫感染。
[Abstract]:experimental purpose
To clone , express and purify the TgACT gene of Toxoplasma gondii , analyze its antigenicity , observe the immune response of mucosal and systemic immune responses induced by different dose of rTgACT immune mice and the ability of the best dose - immunized mice to resist toxoplasmosis infection , and explore the possibility of TgACT as candidate antigen for toxoplasmosis vaccine .
experimental method
In the first part , the recombinant plasmid pET30a - TgACT was constructed and expressed efficiently in E . coli . The recombinant plasmid pET30a - TgACT was purified and purified , the total RNA was extracted , the synthetic primer was designed and the EcoRI and Xho I restriction sites were introduced , and the gene fragment encoding TgActin was cloned into the prokaryotic plasmid pET30a , and the positive clones were identified by double digestion , PCR and sequencing ;
The expression product was induced by IPTG in E . coli BL21 / DE3 . The expression products were identified by SDS - PAGE . The protein was purified by means of dilution and reproducibility . The antigenicity of the protein was purified by using the rabbit anti - Toxoplasma gondii serum .
In the second part , BALB / c mice were randomly divided into 5 groups . The BALB / c mice were randomly divided into 5 groups . The immune groups were immunized with 10 渭g , 20 渭g , 30 渭g , 40 渭grTgACT / L . The levels of serum IgG , IL - 2 and IL - 4 ( 24h ) and IFN - 纬 ( 96h ) were determined by ELISA . The optimal immune dosage of rTgACT was determined .
In the third part , the protective effects of 30 渭rTgACT on mice were observed . BALB / c mice were randomly divided into 2 groups . 30 渭g rTgACT was dissolved in 20 渭l PBS for 3 times . The first and second immunization days were 2 weeks , and the control group received the same amount of PBS . At the 14th day after the last immunization , the mice were killed at 8 脳 104 tachyzoites / rats .
experimental results
In the first part , 1131bp TgACT gene fragment was amplified from the cDNA of RH strain of Toxoplasma gondii , and the recombinant plasmid pET30a - TgACT was constructed successfully . The results showed that the target gene was highly expressed in the form of inclusion bodies in E . coli BL21 / DE3 .
In the second part , the specific immune responses of mice were induced in mice with different doses of rTgACT . The specific IgG , 30 渭g , 40 渭g group were significantly different from those in the control group . There was no significant difference between the specific IgA30渭g group and the control group ( P0.05 ) . There was no significant difference between the groups of IL - 2 .
IL - 4 , 10 gg , 20 渭g , 30 渭g , 40 渭g increased ( P0.05 ) , and there was no significant difference between the immune groups .
IFN - 纬 , 10 渭g , 20 渭g , 30 渭g , 40 渭g increased significantly ( P0.05 ) . In all , 20 渭g , 30 渭g , 40 渭g could induce immune response , and 30 渭g group could induce mucosal and systemic immune response more effectively .
The third part : 30 渭g rTgACT and PBS were injected into the mice . After the last immunization for 2 weeks , the mice were killed in 10 rats and the immune group died 10 . There were 77.89 卤 20.00 脳 105 / g in the immune group and 77.89 卤 20.00 脳 105 / g in the immune group .
The rate of worm reduction in brain tissue was 13.20 卤 2.54 脳 105 / g , which was significantly lower than that in control group ( 22.29 卤 3.35 脳 105 / g ( P0.05 ) .
Conclusion
The recombinant plasmid pET30a - TgACT was successfully constructed and highly expressed in E . coli . rTgACT had an antigenicity of 30 渭g rTgACT , which induced mucosal and systemic immune responses and was effective against Toxoplasma gondii infection .
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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