Mostaan Saied, Ghasemzadeh Abbas, Ehsani Parastoo, Sardari Soroush, Shokrgozar Mohammad Ali, Abolhassani Mohsen, Brujeni Gholamreza Nikbakht
Department of Molecular Biology, Pasteur Institute of Iran, Tehran, Iran.
Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
Adv Biomed Res. 2020 Aug 28;9:43. doi: 10.4103/abr.abr_245_19. eCollection 2020.
is the causative agent of many diseases. Antimicrobial treatment disadvantages highlight the need to find other possible ways such as prophylaxis to manage infections. Current vaccines against this agent include inactivated bacteria, live-attenuated bacteria, and nonpathogenic bacteria, which have disadvantages such as lack of immunogenicity, reactogenicity, or reversion to virulence wild bacteria. Using bioinformatical approaches, potentially immunogenic and protective epitopes identified and merged to design the best epitope fusion form in case of immunogenicity as a vaccine candidate.
In this study, the fusion protein () and full genes () were cloned in pET28a in BL21 (DE3) firstly and later in pBAD/gIII A and expressed in Top10 coli. Overlap polymerase chain reaction (PCR) using different primers for 5' and 3' end of each segment produced fusion segment 1 + 2 and (1 + 2) +3 fragments and was used for cloning.
Cloning of both and into the pET28a vector and their transform into the BL21 (DE3) host was successful, as the presence of the cassettes was proved by digestion and colony PCR, however, their expression faced some challenges independent of expression inducer (isopropyl β-d-1-thiogalactopyranoside) concentration.
Changing the vector to pBAD/gIII A and consequently changing the host to Top10 have resulted in sufficient expression, which shows that Top10 may be a good substitute for such cases. Furthermore, it is concluded that adding 8M urea results in sufficient purification, which hypothesizes that denature purification is better for such cases than native one. Purified proteins headed for further analysis as vaccine candidates.
是多种疾病的病原体。抗菌治疗的缺点凸显了寻找其他可能方法（如预防）来控制感染的必要性。目前针对该病原体的疫苗包括灭活细菌、减毒活细菌和非致病细菌，这些疫苗存在缺乏免疫原性、反应原性或回复至野生型细菌毒力等缺点。利用生物信息学方法，识别并合并潜在的免疫原性和保护性表位，以设计出具有免疫原性的最佳表位融合形式作为候选疫苗。
在本研究中，融合蛋白（）和全长基因（）首先克隆到BL21（DE3）中的pET28a载体中，随后克隆到pBAD/gIII A中并在Top10大肠杆菌中表达。使用针对每个片段5'和3'端的不同引物进行重叠聚合酶链反应（PCR），产生融合片段1 + 2和（1 + 2）+3片段，并用于克隆。
将和克隆到pET28a载体并转化到BL21（DE3）宿主中均成功，通过酶切和菌落PCR证明了盒式结构的存在，然而，它们的表达面临一些挑战，且与表达诱导剂（异丙基β - d - 1 - 硫代半乳糖苷）浓度无关。
将载体更换为pBAD/gIII A并相应地将宿主更换为Top10已实现充分表达，这表明Top10可能是此类情况的良好替代物。此外，得出结论，添加8M尿素可实现充分纯化，推测变性纯化在此类情况下比天然纯化更好。纯化后的蛋白质将作为候选疫苗进行进一步分析。
