Cloning and Production of Antigen 85A Mycobacterium tuberculosis for Diagnostic Latent Tuberculosis: a Preliminary Study

Q3 Agricultural and Biological Sciences

HAYATI Journal of Biosciences Pub Date : 2023-12-06 DOI:10.4308/hjb.31.2.374-381

R. Agus, Sjafaraenan, Yulianti Samara, Muhammad Nasrum Massi

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Abstract

The main challenge in the management of Tuberculosis (TB) is diagnosing quickly and accurately, especially Latent Tuberculosis Infection (LTBI). LTBI detection was carried out using the Tuberculin Skin Test (TST) and Interferon Gamma Release Assay (IGRA). In TB endemic areas, these two examinations have limitations, so current research is directed at finding specific antigens for the diagnosis of LTBI. One of the potential proteins is Antigen 85A (Ag85A) Mycobacterium tuberculosis (Mtb) encoded by Fibronectin-binding protein A (FbpA). The Ag85 complex induces the proliferation of T-cells and interferon-gamma in most healthy individuals infected with Mycobacterium tuberculosis, Mycobacterium leprae, and BCG-vaccinated mice, making it a potential antigen. This study aims to clone and produce recombinant protein Ag85A from Mtb in Escherichia coli BL21. The methods used were ligation to the pET-32a expression vector, transformation to Escherichia coli BL21, and production of protein by IPTG induction. Characterization of recombinant clones by colony PCR and sequencing. The results obtained were that the fbpA gene isolated from Mtb clinical isolate had been amplified, and the PCR product was 900 bp. The production of Antigen 85A has been successfully carried out and produces 44 kDa.

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用于诊断潜伏肺结核的 85A 结核分枝杆菌抗原的克隆和生产：初步研究

肺结核（TB）治疗的主要挑战在于快速准确的诊断，尤其是潜伏肺结核感染（LTBI）。对潜伏肺结核感染的检测采用结核菌素皮肤试验（TST）和干扰素γ释放测定（IGRA）。在结核病流行地区，这两种检查方法都有局限性，因此目前的研究方向是寻找诊断 LTBI 的特异性抗原。由纤维连接蛋白结合蛋白 A（FbpA）编码的结核分枝杆菌（Mtb）抗原 85A（Ag85A）就是其中一种潜在的蛋白。Ag85 复合物可诱导大多数感染结核分枝杆菌、麻风分枝杆菌的健康人和接种卡介苗的小鼠体内的 T 细胞增殖和γ干扰素，因此是一种潜在的抗原。本研究的目的是在大肠杆菌 BL21 中克隆和生产来自 Mtb 的重组蛋白 Ag85A。采用的方法是连接到 pET-32a 表达载体，转化到大肠杆菌 BL21，并通过 IPTG 诱导生产蛋白。通过菌落 PCR 和测序鉴定重组克隆。结果显示，从 Mtb 临床分离株中分离出的 fbpA 基因已被扩增，PCR 产物为 900 bp。抗原 85A 的生产已成功进行，并产生了 44 kDa 的抗原。

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来源期刊

HAYATI Journal of Biosciences Agricultural and Biological Sciences-Agricultural and Biological Sciences (all)

CiteScore

1.10

自引率

0.00%

发文量

审稿时长

24 weeks

期刊介绍： HAYATI Journal of Biosciences (HAYATI J Biosci) is an international peer-reviewed and open access journal that publishes significant and important research from all area of biosciences fields such as biodiversity, biosystematics, ecology, physiology, behavior, genetics and biotechnology. All life forms, ranging from microbes, fungi, plants, animals, and human, including virus, are covered by HAYATI J Biosci. HAYATI J Biosci published by Department of Biology, Bogor Agricultural University, Indonesia and the Indonesian Society for Biology. We accept submission from all over the world. Our Editorial Board members are prominent and active international researchers in biosciences fields who ensure efficient, fair, and constructive peer-review process. All accepted articles will be published on payment of an article-processing charge, and will be freely available to all readers with worldwide visibility and coverage.