Fields H A, Khudyakov Y E, Favorov M O, Khudyakova N S, Cong M E, Holloway B F, Lambert S B, Jue D L
Hepatitis Branch, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Public Health Service, US Department of Health and Human Services, 1600 Clifton Rd., Atlanta, GA 30333, USA.
Clin Diagn Virol. 1996 May;5(2-3):167-79. doi: 10.1016/0928-0197(96)00218-8.
Naturally occurring viral proteins derived from cell culture and recombinant proteins expressed in procaryotic systems have been used extensively as target proteins in the development of immunoassay methods for the detection of antibodies. However, immunoassays utilizing these proteins often yield false-positive reactions suggesting that it may be possible to identify and remove regions responsible for these non-specific reactions.
In this paper we describe a new strategy for the construction of immunoreactive recombinant proteins designed to improve immunoassay specificity.
A synthetic gene encoding an artificial polypeptide composed of antigenic epitopes of the hepatitis E virus (HEV) proteins was constructed from short oligonucleotides by the polymerase chain reaction (PCR). The polypeptide comprises a mosaic of three antigenically dominant regions from the protein encoded by open reading frame 2 (ORF2), one antigenically active region from the protein encoded by ORF3 of the Burmese HEV strain, and one antigenically active region from the protein encoded by ORF3 of the Mexican strain. The mosaic protein was expressed in Escherichia coli as a chimera with glutathione-S-transferase or beta-galactosidase.
Guinea pig sera containing antibodies to the corresponding HEV synthetic peptides were used to demonstrate by immunoblot analysis and by enzyme immunoassay (EIA) the presence and accessibility of all HEV-specific antigenic epitopes designed into the mosaic protein. Both hybrid proteins were shown by immunoblot analysis using a panel of human anti-HEV-positive and -negative sera to be HEV-specific. A sensitive and specific EIA was developed to detect IgG anti-HEV activity in human sera. A neutralization test using individual synthetic peptides corresponding to the epitopes designed into the mosaic protein was also developed to confirm IgG anti-HEV activity by absorbing the specimen before retesting by EIA.
An artificial mosaic protein composed of short linear HEV-specific antigenic epitopes was constructed from synthetic oligonucleotides by PCR and used to develop a sensitive and specific EIA for the detection of anti-HEV activity in human sera.
源自细胞培养的天然病毒蛋白以及在原核系统中表达的重组蛋白已被广泛用作检测抗体的免疫测定方法开发中的靶蛋白。然而，利用这些蛋白的免疫测定常常产生假阳性反应，这表明有可能识别并去除导致这些非特异性反应的区域。
在本文中，我们描述了一种构建免疫反应性重组蛋白的新策略，旨在提高免疫测定的特异性。
通过聚合酶链反应（PCR）从短寡核苷酸构建了一个合成基因，该基因编码由戊型肝炎病毒（HEV）蛋白的抗原表位组成的人工多肽。该多肽由开放阅读框2（ORF2）编码的蛋白的三个抗原显性区域、缅甸HEV株ORF3编码的蛋白的一个抗原活性区域以及墨西哥株ORF3编码的蛋白的一个抗原活性区域组成。该嵌合蛋白在大肠杆菌中作为与谷胱甘肽-S-转移酶或β-半乳糖苷酶的嵌合体表达。
含有针对相应HEV合成肽抗体的豚鼠血清用于通过免疫印迹分析和酶免疫测定（EIA）证明设计到嵌合蛋白中的所有HEV特异性抗原表位的存在和可及性。使用一组人抗HEV阳性和阴性血清进行的免疫印迹分析表明，这两种杂合蛋白都是HEV特异性的。开发了一种灵敏且特异的EIA来检测人血清中的IgG抗HEV活性。还开发了一种使用与设计到嵌合蛋白中的表位相对应的单个合成肽的中和试验，通过在EIA重新检测之前吸收标本以确认IgG抗HEV活性。
通过PCR从合成寡核苷酸构建了由短线性HEV特异性抗原表位组成的人工嵌合蛋白，并用于开发一种灵敏且特异的EIA，以检测人血清中的抗HEV活性。
