前纤维蛋白多克隆抗体对刚地弓形虫在小鼠胚胎成纤维细胞中增殖的影响

doi:10.12140/j.issn.1000-7423.2019.04.012

摘要/Abstract

摘要：

目的制备抗刚地弓形虫前纤维蛋白（TgPRF）的多克隆抗体,初步研究其对刚地弓形虫在小鼠胚胎成纤维细胞中增殖的影响。方法提取刚地弓形虫RH株RNA、扩增TgPRF基因,并构建pET-30a(+)-TgPRF重组质粒,将其转化至大肠埃希菌BL21(DE3)感受态细胞,用异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）鉴定蛋白表达情况。目的蛋白经AKTA系统进行亲和层析纯化和超滤浓缩后进行蛋白质印迹分析（Western blotting）。将2 mg纯化的重组蛋白TgPRF与等体积弗氏完全佐剂混合乳化后,背部皮下多点注射免疫新西兰兔2只,首次免疫后每隔2周,用1 mg纯化的重组蛋白TgPRF与等体积弗氏不完全佐剂加强免疫3次,末次免疫后10 d心脏采血。免疫后血清进行Western blotting鉴定及ELISA抗体效价检测。将胚胎成纤维细胞接种于96孔板中（4 × 10³个细胞/孔）,并加入10⁴个刚地弓形虫速殖子(RH-GFP)。设空白对照组（A组）,抗体干预组（B1、B2和B3组）,阴性血清组（C组）。B1、B2和B3组分别加入1 : 20、1 : 80、1 : 200稀释的抗TgPRF兔血清100 μl;C组加入1 : 20稀释的免疫前兔血清100 μl,每组均设3个复孔。培养72 h后荧光显微镜观察,通过Image pro 6.0软件分析虫体增殖情况。采用SPSS13.0软件进行统计学分析。结果 PCR扩增TgPRF基因片段为510 bp,pET-30a(+)-TgPRF经测序与目的序列完全一致。经IPTG诱导表达,TgPRF蛋白相对分子质量（M_r）约为25 000,且高表达条带主要存在于上清中。TgPRF蛋白经纯化浓缩后浓度为4 mg/ml,Western blotting分析显示,在M_r 25 000处出现特异性条带。经细胞计数试剂盒（CCK-8法）检测,抗TgPRF血清对胚胎成纤维细胞未见毒性损伤。ELISA检测抗TgPRF血清抗体效价 > 1 : 10⁵;各实验组细胞于感染24 h时,A组（0.62 ± 0.23）及C组（0.74 ± 0.25）相对虫体数量高于B1组(0.35 ± 0.16)（P < 0.05）,但与B2和B3组间差异无统计学意义（P > 0.05）,B1~B3组虫体增殖抑制率分别为43.5%、11.4%、3.6%。感染48 h时,A组(3.61 ± 0.66)与C组（3.38 ± 0.78）相对虫体数量明显高于B1~B3组（P < 0.01）,而B1组（1.09 ± 0.58）与B2组（1.92 ± 0.73）低于B3组（2.47 ± 0.84）（P < 0.05）,B1~B3组虫体增殖抑制率分别增高至69.9%、46.9%、31.7%;感染72 h时,A组（19.90 ± 3.92）与C组（20.61 ± 4.07）相对虫体数量高于B1组（5.58 ± 2.43）与B2组（8.06 ± 2.66）（P < 0.01）,B3组（16.02 ± 6.46）明显升高。B1~B3组抑制率分别为72.0%、59.5%、19.5%。结论弓形虫TgPRF蛋白能有效刺激新西兰兔产生抗体,且TgPRF兔血清抗体在体外能有效抑制弓形虫速殖子在小鼠胚胎成纤维细胞中的增殖并呈现明显的量效关系,但并不能完全消除虫体。

关键词: 刚地弓形虫, 前纤维蛋白, 多克隆抗体, 原核表达

Abstract:

Objective A polyclonal antibody against Toxoplasma gondii profilin (TgPRF) was prepared in two rabbits and its inhibitory effect on the proliferation of T. gondii in mouse embryonic fibroblasts was observed. Methods The total RNA was extracted from T. gondii RH strain and total cDNA was reversely transcribed. The cDNA encoding for TgPRF gene was amplified from the total cDNA and then cloned into prokaryotic expression vector pET-30a(+). The recombinant pET-30a(+)-TgPRF plasmid was transformed into Escherichia coli BL21 (DE3) and the recombinant TgPRF protein was expressed under IPTG induction. The expressed recombinant protein with His-tag was purified by affinity chromatography with AKTA system and the purified recombinant TgPRF was analyzed by SDS-PAGE and Western blotting. Two New Zealand rabbits were each immunized with 1 mg recombinant TgPRF emulsified with the same volume of Freund’s complete adjuvant by multiple subcutaneous injections on the back, then boosted three times with the same amount of protein formulated with incomplete adjuvant with two weeks interval. Blood was collected from rabbit heart 10 days after the last immunization. The anti-TgPRF specific antibody in the post-immune serum was identified by Western blotting and titrated by ELISA. Mouse embryonic fibroblasts were seeded in 96-well plates (4 × 10³ cells/well) and then infected with 10⁴ T. gondii tachyzoites (RH-GFP). The inhibition of T. gondii tachyzoites in infected embryonic fibroblasts by anti-TgPRF serum was observed when anti-TgPRF serum was added at 1 : 20 (B1), 1 : 80 （B2） and 1 : 200 （B3） dilutions. The pre-immune serum at 1 : 20 was added as negative control (C) and blank medium was used as blank control(A). Each sample was added in triplicate and cultured for 72 h, then observed under fluorescence microscope. Proliferation of tachyzoites was analyzed by Image pro 6.0 software. Results The cDNA encoding for TgPRF was amplified from T. gondii total cDNA by PCR with size of 510 bp, and then subcloned into pET-30a(+) for expression in E. coli. The cloned recombinant pET-30a-TgPRF plasmid contained the correct target sequence confirmed by DNA sequencing. The recombinant TgPRF protein was successfully expressed in E. coli BL21 as size of 25 000 mainly in soluble supernatant fraction. After being purified and concentrated, the concentration of recombinant TgPRF protein was determined by BCA as 4 mg/ml. The expressed recombinant TgPRF was used to immunize two rabbits to obtain antiserum and the recombinant TgPRF was recognized by the rabbit anti-TgPRF serum determined by Western blotting analysis. The rabbit anti-TgPRF serum had no toxic effect on embryonic fibroblasts determined by CCK-8 assay. The anti-TgPRF antibody titer was > 1 : 10⁵ determined by ELISA. After being incubated with anti-TgPRF for 24 h, the T. gondii tachyzoites proliferation was significantly inhibited in group incubated with 1 : 20 antiserum (the relative parasite amount 0.35 ± 0.16) compared with blank control (0.62 ± 0.23) and pre-immune serum control (0.74 ± 0.25) (P < 0.05), however, there was no significant difference among groups incubated with antiserum of 1 : 80, 1 : 200 and blank control and pre-immune serum control (P > 0.05). The tachyzoite proliferation inhibitory rates in group B1-B3 were 43.5%, 11.4%, 3.6% respectively. After being incubated with antiserum for 48 h, the relative tachyzoites amount in group incubated with antiserum of 1 : 20, 1 : 80 and 1 : 200 were 1.09 ± 0.58, 1.92 ± 0.73 and 2.47 ± 0.84, respectively, that were significant lower than blank control group (3.61 ± 0.66) and pre-immune serum group (3.38 ± 0.78) (P < 0.01) with inhibitory rates of 69.9%, 46.9%, and 31.7%, respectively. After being incubated with antiserum for 72 h, the tachyzoites proliferation was significantly inhibited in groups with antiserum at 1 : 20 (5.58 ± 2.43), 1 : 80 (8.06 ± 2.66) (P < 0.01) and 1 : 200 (16.02 ± 6.46) compared with blank control (19.90 ± 3.92) and pre-immune serum control (20.61 ± 4.07), with inhibitory rates of 72.0%, 59.5%, and 19.5%, respectively. Conclusion The E. coli expressed recombinant TgPRF protein was immunogenic and induced strong antibody response in immunized rabbits. The rabbit anti-TgPRF serum could effectively inhibit the proliferation of T. gondii tachyzoites in mouse embryonic fibroblast cells at a dose-dependent pattern in vitro, but could not completely eliminate the parasite.

Key words: Toxoplasma gondii, Profilin, Polyclonal antibody, Prokaryotic expression

中图分类号:

R382.5

杨瑞, 熊乙丁, 郭珊珊, 廖业, 夏嫱, 郭锦锦, 杜联峰. 前纤维蛋白多克隆抗体对刚地弓形虫在小鼠胚胎成纤维细胞中增殖的影响[J]. 中国寄生虫学与寄生虫病杂志, 2019, 37(4): 437-443.

Rui YANG, Yi-ding XIONG, Shan-shan GUO, Ye LIAO, Qiang XIA, Jin-jin GUO, Lian-feng DU. Inhibitory effect of anti-profilin antibody on the proliferation of Toxoplasma gondii in mouse embryonic fibroblasts[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2019, 37(4): 437-443.

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