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首页> 外文期刊>Journal of Veterinary Research >In vitro evaluation of chitosan-DNA plasmid complex encoding Jembrana disease virus Env-TM protein as a vaccine candidate
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In vitro evaluation of chitosan-DNA plasmid complex encoding Jembrana disease virus Env-TM protein as a vaccine candidate

机译：壳聚糖-DNA质粒复合体的体外评价编码Jembrana病毒Env-TM蛋白作为疫苗候选者

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Introduction: The development of Jembrana disease vaccine is an important effort to prevent losses in the Bali cattle industry in Indonesia. This study aims to prepare a Jembrana DNA vaccine encoding the transmembrane portion of the envelope protein in pEGFP-C1 and test the success of its delivery in culture cells using a chitosan-DNA complex. Material and Methods: Cloning of the DNA vaccine was successfully performed on E. coli DH5 alpha and confirmed by colony PCR, restriction analysis and sequencing. The plasmids were prepared as a chitosan complex using the complex coacervation method and physicochemically characterised using a particle size analyser. A transfection assay was performed in HeLa cells with 4 h exposure, and mRNA expression was assessed at 24 h post transfection. Results: With a 1:2 (wt./wt.) ratio of DNA and chitosan, the complexes have a mean diameter of 236 nm, zeta potential value of + 17.9 mV, and showed no high toxicity potential in the HeLa cells. This complex successfully delivered the DNA into cells, as shown by the presence of a specific RT-PCR product (336 bp). However, the real-time PCR analysis showed that the delivery with chitosan complex resulted in lower target mRNA expression when compared with a commercial transfecting agent. Conclusion: pEGFP-env-tm JDV as a candidate vaccine can be delivered as the chitosan-DNA complex and be expressed at the transcription level in vitro. This initial study will be used for further improvement and evaluation in vivo.
机译：简介：Jembrana病疫苗的发展是防止印度尼西亚巴厘岛行业损失的重要努力。本研究旨在制备在PEGFP-C1中编码包络蛋白的跨膜部分的Jembrana DNA疫苗，并使用壳聚糖-DNA复合物测试其在培养细胞中的递送的成功。材料和方法：在大肠杆菌DH5α上成功进行DNA疫苗的克隆，并通过菌落PCR，限制性分析和测序证实。使用络合物凝聚方法制备质粒作为壳聚糖络合物，并使用粒度分析仪进行物理化学表征。在HeLa细胞中进行转染测定，具有4小时暴露，并在转染后24小时评估mRNA表达。结果：DNA和壳聚糖的比例为1：2（wt./wt。）的比例，复合物的平均直径为236nm，Zeta电位值+ 17.9mV，并且在HeLa细胞中显示出高毒性电位。该复合物成功地将DNA递送到细胞中，如通过特异性RT-PCR产物（336bp）所示。然而，与商业转染剂相比，实时PCR分析表明用壳聚糖复合物递送导致靶mRNA表达下降。结论：PEGFP-ENV-TM JDV作为候选疫苗可以作为壳聚糖-DNA复合物递送，并在体外转录水平表达。该初步研究将用于体内进一步改进和评估。

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