Immunogenic Response Assessment of Hepatitis Delta Virus Antigen from Pakistani Isolate in Rabbits Using a Prokaryotic Expression System.

IF 1.2 4区 医学 Q4 IMMUNOLOGY
Hifza Manzoor, Muhammad Shahid, Nadeem Ahmed, Samia Afzal, Muhammad Huzaifa, Saad Tahir, Iram Amin
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Abstract

The hepatitis delta virus (HDV) is a defective and small, blood-borne viroid-like pathogen that coinfects with the hepatitis B virus (HBV) in about 5% of the infected individuals as it is a satellite virus of HBV. The treatment of HDV infection is quite challenging because there is no vaccine against HDV. Several commercial PCR and in-house assays have been developed, but there are greater variations in the results of these assays because they are not standardized properly. Studies are also delayed because of the unavailability of commercial HDV-specific antibodies for the diagnosis of HDV infection, even for the research devotions. To fill this gap, the recombinant antigenic HDAg protein of genotype I of HDV from the local isolate was successfully expressed and purified in the Escherichia coli (E. coli) system, followed by anti-HDAg antibodies production in rabbits. After determining and amplifying the antigenic region of HDAg of HDV, the fragment was cloned into the pET-28a vector and expressed in E. coli TOP10 cells. Following the successful expression of recombinant HDAg protein with a His-tag at its C-terminal end, we purified the HDAg protein using affinity chromatography. Both the expression and purification of HDAg-An protein were confirmed through SDS-PAGE and Western blot analysis. Through proper optimization, the HDAg-An protein was obtained with more than 90% purity. The next step involved immunizing Japanese White rabbits with the purified HDAg. The immunization protocol included 80 µg of HDAg-An in two subcutaneous priming doses and four 40 µg booster doses, followed by blood collection two weeks after each boost to monitor antibody production. The level of anti-HDAg antibodies in the rabbit serum was assessed using a quantitative ELISA technique. In the future, these antibodies could be used for the development of HDV-specific in-house assays as well as vaccines against the HDV genotype I that is locally predominant, potentially decreasing the burden of imported diagnostic tools and reagents.

用原核表达系统评价兔巴基斯坦分离物丁型肝炎病毒抗原的免疫原性反应。
丁型肝炎病毒(HDV)是一种有缺陷的小型血源性病毒样病原体,约5%的感染者与乙型肝炎病毒(HBV)合并感染,因为它是乙型肝炎病毒的卫星病毒。由于没有针对HDV的疫苗,治疗HDV感染相当具有挑战性。已经开发了几种商业PCR和内部分析方法,但这些分析的结果差异较大,因为它们没有适当地标准化。由于无法获得用于诊断HDV感染的商用HDV特异性抗体,甚至用于研究投入,研究也被推迟。为了填补这一空白,在大肠杆菌系统中成功表达并纯化了HDV基因I型重组抗原HDAg蛋白,随后在家兔体内产生了抗HDAg抗体。确定并扩增HDV HDAg抗原区后,将片段克隆到pET-28a载体中,在大肠杆菌TOP10细胞中表达。在成功表达重组HDAg蛋白后,我们利用亲和层析纯化了HDAg蛋白。通过SDS-PAGE和Western blot分析证实了HDAg-An蛋白的表达和纯化。通过适当的优化,获得了纯度在90%以上的HDAg-An蛋白。下一步是用纯化的HDAg免疫日本大白兔。免疫方案包括两次皮下注射80µg HDAg-An和四次注射40µg加强剂,每次加强后两周采集血液以监测抗体产生。采用定量ELISA技术检测兔血清中抗hdag抗体水平。在未来,这些抗体可用于开发HDV特异性内部检测以及针对HDV基因I型的疫苗(在当地占主导地位),从而有可能减少进口诊断工具和试剂的负担。
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来源期刊
Viral immunology
Viral immunology 医学-病毒学
CiteScore
3.60
自引率
0.00%
发文量
84
审稿时长
6-12 weeks
期刊介绍: Viral Immunology delivers cutting-edge peer-reviewed research on rare, emerging, and under-studied viruses, with special focus on analyzing mutual relationships between external viruses and internal immunity. Original research, reviews, and commentaries on relevant viruses are presented in clinical, translational, and basic science articles for researchers in multiple disciplines. Viral Immunology coverage includes: Human and animal viral immunology Research and development of viral vaccines, including field trials Immunological characterization of viral components Virus-based immunological diseases, including autoimmune syndromes Pathogenic mechanisms Viral diagnostics Tumor and cancer immunology with virus as the primary factor Viral immunology methods.
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