Department of Reproduction and Obstetrics, Faculty of Veterinary Medicine, University Gadjah Mada, Yogyakarta 55281, Indonesia.
Biomedical Field, Nursing Study Program, STIKES Yarsi Mataram, West Nusa Tenggara 83361, Indonesia.
J Vet Sci. 2021 Nov;22(6):e76. doi: 10.4142/jvs.2021.22.e76. Epub 2021 Aug 30.
The development of a vaccine for Jembrana disease is needed to prevent losses in Indonesia's Bali cattle industry. A DNA vaccine model (pEGFP-C1-tat) that requires a functional delivery system will be developed. Polylactic-co-glycolic acid (PLGA) may have potential as a delivery system for the vaccine model.
This study aims to evaluate the potential of PLGA as a delivery system for pEGFP-C1-tat.
Consensus and codon optimization for the tat gene was completed using a bioinformatic method, and the product was inserted into a pEGFP-C1 vector. Cloning of the pEGFP-C1-tat was successfully performed, and polymerase chain reaction (PCR) and restriction analysis confirmed DNA isolation. PLGA-pEGFP-C1-tat solutions were prepared for encapsulated formulation testing, physicochemical characterization, stability testing with DNase I, and cytotoxicity testing. The PLGA-pEGFP-C1-tat solutions were transfected in HeLa cells, and gene expression was observed by fluorescent microscopy and real-time PCR.
The successful acquisition of transformant bacteria was confirmed by PCR. The PLGA:DNA:polyvinyl alcohol ratio formulation with optimal encapsulation was 4%:0.5%:2%, physicochemical characterization of PLGA revealed a polydispersity index value of 0.246, a particle size of 925 nm, and a zeta potential value of -2.31 mV. PLGA succeeded in protecting pEGFP-C1-tat from enzymatic degradation, and the percentage viability from the cytotoxicity test of PLGA-pEGFP-C1-tat was 98.03%. The PLGA-pEGFP-C1-tat demonstrated luminescence of the EGFP-tat fusion protein and mRNA transcription was detected.
PLGA has good potential as a delivery system for pEGFP-C1-tat.
为了防止印度尼西亚巴厘牛产业的损失，需要开发一种针对杰姆巴兰病的疫苗。将开发一种需要功能性传递系统的 DNA 疫苗模型（pEGFP-C1-tat）。聚乳酸-共-羟基乙酸（PLGA）可能有潜力作为疫苗模型的传递系统。
本研究旨在评估 PLGA 作为 pEGFP-C1-tat 传递系统的潜力。
使用生物信息学方法完成 tat 基因的共识和密码子优化，并将产物插入 pEGFP-C1 载体中。成功克隆了 pEGFP-C1-tat，聚合酶链反应（PCR）和限制分析证实了 DNA 的分离。为封装配方测试、DNase I 稳定性测试和细胞毒性测试制备了 PLGA-pEGFP-C1-tat 溶液。将 PLGA-pEGFP-C1-tat 溶液转染 HeLa 细胞，通过荧光显微镜和实时 PCR 观察基因表达。
通过 PCR 证实了转化菌的成功获得。具有最佳封装的 PLGA:DNA:聚乙烯醇配方为 4%:0.5%:2%，PLGA 的理化特性分析显示多分散指数值为 0.246、粒径为 925nm 和 Zeta 电位值为-2.31mV。PLGA 成功地保护了 pEGFP-C1-tat 免受酶降解，细胞毒性试验中 PLGA-pEGFP-C1-tat 的存活率为 98.03%。PLGA 显示出 EGFP-tat 融合蛋白的发光，并且检测到 mRNA 转录。
PLGA 作为 pEGFP-C1-tat 的传递系统具有良好的潜力。
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