Torque Teno 病毒（TTV）基因组 N22 区的克隆与表达及利用肽段建立 TTV 抗体免疫检测方法的研究。
Cloning and expression of N22 region of Torque Teno virus (TTV) genome and use of peptide in developing immunoassay for TTV antibodies.
机构信息
Clinical Biochemistry Division, Department of Laboratory Medicine, All India Institute of Medical Sciences, New Delhi 110029, India.
出版信息
Virol J. 2014 May 20;11:96. doi: 10.1186/1743-422X-11-96.
BACKGROUND
Torque Teno Virus (TTV) is a DNA virus with high rate of prevalence globally. Since its discovery in 1997, several studies have questioned the role of this virus in causing disease. However, it still remains an enigma. Although methods are available for detection of TTV infection, there is still a need for simple, rapid and reliable method for screening of this virus in human population. Present investigation describes the cloning and expression of N22 region of TTV-genome and the use of expressed peptide in development of immunoassay to detect anti-TTV antibodies in serum. Since TTV genotype-1 is more common in India, the serum positive for genotype-1 was used as source of N22 for expression purpose.
METHODS
Full length N22 region of ORF1 from TTV genotype-1 was amplified and cloned in pGEM®-T Easy vector. After cloning, the amplicon was transformed and expressed as a fusion protein containing hexa-histidine tag in pET-28a(+) vector using BL21 E. coli cells as host. Expression was conducted both in LB medium as well as ZYP-5052 auto-induction medium. The expressed peptide was purified using metal-chelate affinity chromatography and used as antigen in developing a blot immunoassay.
RESULTS
Analysis of translated product by SDS-PAGE and western blotting demonstrated the presence of 25 kDa polypeptide produced after expression. Solubility studies showed the polypeptide to be associated with insoluble fraction. The use of this peptide as antigen in blot assay produced prominent spot on membrane treated with sera from TTV-infected patients. Analysis of sera from 75 patients with liver and renal diseases demonstrated a successful implication of N22 polypeptide based immunoassay in screening sera for anti-TTV antibodies. Comparison of the immunoassay developed using expressed N22 peptide with established PCR method for TTV-DNA detection showed good coherence between TTV-DNA and presence of anti-TTV antibodies in the sera analysed.
CONCLUSIONS
This concludes that TTV N22 region may be expressed and safely used as antigen for blot assay to detect anti-TTV antibodies in sera.
背景
Torque Teno 病毒（TTV）是一种具有全球高流行率的 DNA 病毒。自 1997 年发现以来，已有多项研究质疑该病毒在致病中的作用。然而，它仍然是一个谜。虽然有检测 TTV 感染的方法，但仍需要一种简单、快速和可靠的方法来筛选人群中的这种病毒。本研究描述了 TTV 基因组 N22 区的克隆和表达，并利用表达的肽开发免疫检测法来检测血清中的抗 TTV 抗体。由于 TTV 基因型-1 在印度更为常见，因此用于表达目的的 N22 来源于血清阳性的基因型-1。
方法
从 TTV 基因型-1 的 ORF1 扩增全长 N22 区，并克隆到 pGEM®-T Easy 载体中。克隆后，使用 BL21 E. coli 细胞作为宿主，将扩增子转化并在 pET-28a(+)载体中表达为含有六组氨酸标签的融合蛋白。在 LB 培养基和 ZYP-5052 自动诱导培养基中进行表达。使用金属螯合亲和层析纯化表达的肽，并将其用作开发印迹免疫检测法的抗原。
结果
SDS-PAGE 和 Western blot 分析翻译产物表明，表达后产生了 25 kDa 的多肽。可溶性研究表明该多肽与不溶性部分相关。该肽用作印迹检测法中的抗原，可在与感染 TTV 的患者血清处理过的膜上产生明显的斑点。对 75 例肝肾功能疾病患者血清的分析表明，基于 N22 多肽的免疫检测法成功地用于筛选血清中的抗 TTV 抗体。使用表达的 N22 肽开发的免疫检测法与已建立的 TTV-DNA 检测 PCR 方法的比较表明，在分析的血清中，TTV-DNA 和抗 TTV 抗体的存在之间具有良好的一致性。
结论
这表明 TTV N22 区可以被表达，并安全地用作印迹检测法中的抗原，以检测血清中的抗 TTV 抗体。
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