毕赤酵母GS115中衣壳蛋白L1 HPV 52自组装病毒样颗粒的表达、纯化和表征

IF 3.6 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Journal, genetic engineering & biotechnology Pub Date : 2023-11-20 DOI:10.1186/s43141-023-00571-0
Chindy Nur Rosmeita, Sri Budiarti, Apon Zaenal Mustopa, Ela Novianti, Sri Swasthikawati, Sheila Chairunnisa, Ai Hertati, Maritsa Nurfatwa, Nurlaili Ekawati, Nurhasni Hasan
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引用次数: 0

摘要

背景:由人乳头瘤病毒(HPV)引起的宫颈癌是全球最常见的恶性肿瘤之一。HPV 52是一种高风险的致癌基因型,已被确定为印度尼西亚最普遍的类型。基于病毒样颗粒(VLP)的HPV感染疫苗可能受益于L1衣壳蛋白的自组装VLP。结果:在0.5%甲醇诱导下,重组人乳头状瘤病毒52 L1在毕赤酵母中成功表达。用拷贝数来比较表达水平和稳定性。选择在含有2000 μg/ml Zeocin的固体培养基上存活的菌落,培养表达HPV 52 L1。从所选菌落中提取DNA,用qPCR测定拷贝数。用快速高效液相色谱法纯化HPV 52 L1蛋白。透射电镜(TEM)鉴定证实了VLP的自组装。在无选择性压力培养基条件下连续培养100代后,基因组DNA保持完整,产生的蛋白质相对稳定。但波段强度略低于亲本群体。在拷贝数方面,低拷贝转化导致低表达,但产生了高度稳定的重组克隆。最终,毕赤酵母中表达的L1蛋白可以自组装成VLP。因此,重组人乳头瘤病毒具有稳定的克隆和自组装成VLP的能力。结论:重组L1型HPV 52蛋白在巴氏酵母中成功表达,其大小范围约为55 kDa,具有良好的稳定性。毕赤酵母中表达的L1蛋白成功地自组装了HPV VLPs,从而确立了其作为预防性疫苗的潜在效力。
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Expression, purification, and characterization of self-assembly virus-like particles of capsid protein L1 HPV 52 in Pichia pastoris GS115.

Background: Cervical cancer caused by the human papillomavirus (HPV) is one of the most frequent malignances globally. HPV 52 is a high-risk cancer-causing genotype that has been identified as the most prevalent type in Indonesia. Virus-like particles (VLP)-based vaccinations against HPV infection could benefit from self-assembled VLP of L1 capsid protein.

Result: The recombinant HPV 52 L1 was expressed in Pichia pastoris on a shake-flask scale with 0.5% methanol induction in this study. The copy number was used to compare the expression level and stability. The colony that survived on a solid medium containing 2000 μg/ml of Zeocin was selected and cultured to express HPV 52 L1. DNA was extracted from the chosen colony, and the copy was determined using qPCR. HPV 52 L1 protein was then purified through fast performance liquid chromatography. Transmission electron microscopy (TEM) evaluation confirmed the VLP self-assembly. The genomic DNA remained intact after 100 generations of serial cultivation under no selective pressure medium conditions, and the protein produced was relatively stable. However, the band intensity was slightly lower than in the parental colony. In terms of copy number, a low copy transformant resulted in low expression but produced a highly stable recombinant clone. Eventually, the L1 protein expressed in Pichia pastoris can self-assemble into VLP. Therefore, recombinant HPV possesses a stable clone and the ability to self-assemble into VLP.

Conclusion: The recombinant L1 HPV 52 protein is successfully expressed in P. pastoris within a size range of approximately 55 kDa and demonstrated favorable stability. The L1 protein expressed in Pichia pastoris successful self-assembled of HPV VLPs, thereby establishing their potential efficacy as a prophylactic vaccine.

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