Optimization of Expression and Extraction of Toxocara canis Recombinant C-Type Lectin Protein in Escherichia coli BL21 (DE3)

IF 1.5 3区 农林科学 Q2 VETERINARY SCIENCES Onderstepoort Journal of Veterinary Research Pub Date : 2021-06-22 DOI:10.22059/JVR.2020.287488.2966
Mahsa Shahbakhsh, F. Jalousian, Seyed Hossein Hosseini, P. Shayan, A. Moghadasi
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Abstract

BACKGROUND: Toxocaiasis is an important zoonotic disease caused by the second stage larvae of Toxocara canis and Toxocara cati. Toxocariasis is the most common worm infection in several temperate countries and causes severe complications. C-type lectin is one of the larval stage products of this parasite. It is involved in immune responses, including cellular signals in vertebrate immunity, activation of innate immunity in vertebrates and non-vertebrates, and induction of homeostasis.  OBJECTIVES: The current research aimed to optimize the production of recombinant C-type lectin of Toxocara canis and investigate its antigenic properties. METHODS: Reference nucleotide sequence of lectin type C (CTL) of Toxocara canis (T. canis) was extracted from Genbank. Recombinant Plasmid, pET32a, including the desired sequence was then constructed by GENERAY. The recombinant plasmid was transformed to Escherichia coli strain BL21 (DE3). The expression of the recombinant protein was investigated using SDS-PAGE and dot blot methods and approved with human T. canis positive serum. RESULTS: The findings of the present study showed that optimization and high level production of recombinant protein expression was achieved by selecting Escherichia coli (BL21 (DE3), 37 °C temperature for 4 hours after induction and 1 mM IPTG concentration. After optimization, the recombinant protein was obtained at a concentration of 1160±0.6 µg/mL. The molecular weight of the resulting recombinant protein was 42 kDa. Recombinant plasmid passage in Escherichia coli DH5α strain caused a significant increase in recombinant protein expression. The results of condition optimization evaluation, with SDS-PAGE and Dot blot methods, showed that the highest production of recombinant C type lectin protein of T. canis was obtained under optimized conditions. CONCLUSIONS: Based on these results, to produce reasonable amounts of specific C-type lectin recombinant protein, further studies are needed to evaluate its immunogenicity and protection against Toxocara canis infection.
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重组犬弓形虫c型凝集素蛋白在大肠杆菌BL21 (DE3)中的表达及提取优化
背景:弓形虫病是由犬弓形虫和猫弓形虫的第二阶段幼虫引起的一种重要的人畜共患疾病。弓形虫病是几个温带国家最常见的蠕虫感染,可引起严重并发症。c型凝集素是这种寄生虫的幼虫期产物之一。它参与免疫反应,包括脊椎动物免疫中的细胞信号,脊椎动物和非脊椎动物先天免疫的激活,以及体内平衡的诱导。目的:优化重组犬弓形虫c型凝集素的生产工艺,研究其抗原性。方法:从Genbank中提取犬弓形虫(T. canis)凝集素C型(CTL)参考核苷酸序列。然后用GENERAY构建重组质粒pET32a,包括所需的序列。将重组质粒转化到大肠杆菌BL21 (DE3)中。利用SDS-PAGE和dot blot方法检测重组蛋白的表达,并与人犬t型虫阳性血清进行了鉴定。结果:本研究结果表明,选择大肠杆菌BL21 (DE3),诱导后温度37℃,诱导后4小时,IPTG浓度为1 mM,实现了重组蛋白的优化和高水平表达。优化后得到的重组蛋白浓度为1160±0.6µg/mL。重组蛋白分子量为42 kDa。重组质粒在大肠杆菌DH5α中传代后,重组蛋白的表达量显著增加。采用SDS-PAGE和Dot blot方法对条件进行优化评价,结果表明,在优化条件下,重组犬犬C型凝集素蛋白的产量最高。结论:在此基础上,为获得合理数量的特异性c型凝集素重组蛋白,需进一步研究其免疫原性及对犬弓形虫感染的保护作用。
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来源期刊
CiteScore
4.30
自引率
0.00%
发文量
13
审稿时长
16 weeks
期刊介绍: The Onderstepoort Journal of Veterinary Research, is the official publication of the Onderstepoort Veterinary Institute. While it considers submissions from any geographic region, its focus is on Africa and the infectious and parasitic diseases and disease vectors that affect livestock and wildlife on the continent.
期刊最新文献
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