Expression, purification, and evaluation of recombinant LecA as a candidate for an amebic colitis vaccine.
Expression, purification, and evaluation of recombinant LecA as a candidate for an amebic colitis vaccine.
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DOI：
10.1016/j.vaccine.2013.06.056
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发表时间：
2014-02-26
期刊：
影响因子：
5.5
通讯作者：
Lyerly, D.
中科院分区：
文献类型：
--
作者：
Barroso, L.;Abhyankar, M.;Noor, Z.;Read, K.;Pedersen, K.;White, R.;Fox, C.;Petri, W. A., Jr.;Lyerly, D.
溶组织内阿米巴可引起阿米巴结肠炎和肝脓肿，被认为是该病流行的发展中国家居民和旅行者的主要肠道病原体。这种原虫与肠道的相互作用是通过滋养体阶段通过半乳糖和N-乙酰-d-氨基半乳糖(Gal/GalNAc)凝集素与肠道粘蛋白和上皮细胞结合而实现的，凝集素由二硫键连接的重链(约180 kDa)和轻链(约35 kDa)和一个非共价结合的中间亚单位(约150 kDa)组成。我们努力开发一种针对这种病原体的疫苗，重点是位于重链亚基半胱氨酸富集区的一个内部578个氨基酸片段，命名为LecA，因为：(I)它是血清阳性个体黏附阻断抗体的主要靶点，(Ii)His标记的LecA疫苗在动物模型中提供保护。我们开发了一种利用在大肠杆菌中表达的密码子优化的基因来制备高纯度非标记LecA的纯化工艺。这一过程包括：(I)细胞裂解、包涵体收集和洗涤；(Ii)变性LecA的溶解和复性；(Iii)抛光凝胶过滤步骤。纯化的片段主要以β-Sheet结构的无规卷曲形式存在，内毒素和核酸含量低，免疫活性高，可诱导识别天然凝集素的抗体，并在体外抑制滋养体与CHO细胞的黏附。用LecA免疫CBA小鼠，对盲肠结肠炎有明显的保护作用。我们的程序产生了足够数量的高纯度的LecA，用于未来对稳定性、免疫原性和蛋白质佐剂配方的保护的研究。
Entamoeba histolytica, which causes amebic colitis and liver abscess, is considered a major enteric pathogen in residents and travelers to developing countries where the disease is endemic. Interaction of this protozoan parasite with the intestine is mediated through the binding of the trophozoite stage to intestinal mucin and epithelium via a galactose and N-acetyl-d-galactosamine (Gal/GalNAc) lectin comprised of a disulfide linked heavy (ca. 180 kDa) and light chain (ca. 35 kDa) and a noncovalently bound intermediate subunit (ca. 150 kDa). Our efforts to develop a vaccine against this pathogen have focused on an internal 578 amino acid fragment, designated LecA, located within the cysteine-rich region of the heavy chain subunit because: (i) it is a major target of adherence-blocking antibodies of seropositive individuals and (ii) vaccination with his-tagged LecA provides protection in animal models. We developed a purification process for preparing highly purified non-tagged LecA using a codon-optimized gene expressed in Escherichia coli. The process consisted of: (i) cell lysis, collection and washing of inclusion bodies; (ii) solubilization and refolding of denatured LecA; and (iii) a polishing gel filtration step. The purified fragment existed primarily as a random coil with β-sheet structure, contained low endotoxin and nucleic acid, was highly immunoreactive, and elicited antibodies that recognized native lectin and that inhibited in vitro adherence of trophozoites to CHO cells. Immunization of CBA mice with LecA resulted in significant protection against cecal colitis. Our procedure yields sufficient amounts of highly purified LecA for future studies on stability, immunogenicity, and protection with protein-adjuvant formulations.
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影响因子：
2.2
作者：
Mann, BJ;Lockhart, LA
通讯作者：
Lockhart, LA
影响因子：
3.1
作者：
Ivory, Catherine P. A.;Chadee, Kris
通讯作者：
Chadee, Kris
影响因子：
2.2
作者：
DIAMOND, LS;CLARK, CG
通讯作者：
CLARK, CG
影响因子：
1.6
作者：
Cash, Heather L.;Whitham, Cecilia V.;Hooper, Lora V.
通讯作者：
Hooper, Lora V.
DOI：
10.4269/ajtmh.1996.55.693
发表时间：
1996-12-01
影响因子：
3.3
作者：
Braga, LL;Lima, AAM;Mann, BJ
通讯作者：
Mann, BJ