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重组人嗜T淋巴细胞病毒I型/II型线性表位抗原的表达、纯化及其在疑似患者筛查中的应用

Expression and purification of recombinant HTLV-I/-II linear epitopes antigen and its application for screening of suspected patients.

作者信息

Faramarzi Roxana, Dolatabadi Samaneh

机构信息

Department of Microbiology, Neyshabur Branch, Islamic Azad University, Neyshabur, Khorasan Razavi, Iran.

出版信息

Iran J Microbiol. 2017 Feb;9(1):43-49.

PMID:28775823
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC5534004/
Abstract

BACKGROUND AND OBJECTIVES

The linear epitopes of gp46-I, gp46-II, gp21 and p19 are used in diagnosis of HTLV-I/-II infections. The aims of this study was to obtain high-level expression and purification of recombinant antigen (RA) containing these epitopes. Large-scale preparation of such antigen probably worths for diagnostic purpose.

MATERIALS AND METHODS

The synthetic DNA encoding RA was synthesized and over-expressed as soluble in BL21 (DE3) cells. Expression and distribution of the His-GST-RA protein were evaluated using SDS-PAGE. The soluble RA was purified utilizing Ni-NTA agarose beads under native conditions and was concentrated by ultra filtration. Using 20 sera specimens from HTLV infected patients, the antigenicity of the purified protein was confirmed in ELISA and western blotting analysis.

RESULTS

SDS-PAGE revealed that the purified protein was more than 90% pure. The final yield was approximately 25 mg per liter of culture medium. ELISA results showed that RA could specifically bind to anti-HTLV-I/-II antibodies in infected sera.

CONCLUSION

RA could be a candidate for HTLV-I/-II screening and the strategy presented in this study could be used for easy production of this diagnostic protein.

摘要

背景与目的

gp46-I、gp46-II、gp21和p19的线性表位用于人类嗜T淋巴细胞病毒I型/II型（HTLV-I/-II）感染的诊断。本研究的目的是获得含这些表位的重组抗原（RA）的高效表达与纯化。大规模制备此类抗原可能对诊断有价值。

材料与方法

合成编码RA的DNA，并在BL21（DE3）细胞中实现可溶性的过表达。采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）评估His-GST-RA蛋白的表达及分布情况。在天然条件下利用镍-亚氨基二乙酸（Ni-NTA）琼脂糖珠对可溶性RA进行纯化，并通过超滤进行浓缩。使用来自HTLV感染患者的20份血清标本，通过酶联免疫吸附测定（ELISA）和蛋白质印迹分析确认纯化蛋白的抗原性。

结果

SDS-PAGE显示纯化后的蛋白纯度超过90%。最终产量约为每升培养基25毫克。ELISA结果表明，RA可与感染血清中的抗HTLV-I/-II抗体特异性结合。

结论

RA可作为HTLV-I/-II筛查的候选物，本研究中提出的策略可用于简便生产这种诊断蛋白。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f975/5534004/e712e45de46b/IJM-9-43-g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f975/5534004/10d62436180d/IJM-9-43-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f975/5534004/e3f07109e201/IJM-9-43-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f975/5534004/063d557aa148/IJM-9-43-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f975/5534004/6f27b93b714c/IJM-9-43-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f975/5534004/7d2b5ff0766d/IJM-9-43-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f975/5534004/e712e45de46b/IJM-9-43-g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f975/5534004/10d62436180d/IJM-9-43-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f975/5534004/e3f07109e201/IJM-9-43-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f975/5534004/063d557aa148/IJM-9-43-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f975/5534004/6f27b93b714c/IJM-9-43-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f975/5534004/7d2b5ff0766d/IJM-9-43-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/f975/5534004/e712e45de46b/IJM-9-43-g006.jpg

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