Department of Biology, Marvdasht Branch, Islamic Azad University, Marvdasht, Iran.
Department of Biology, Damghan Branch, Islamic Azad University, Damghan, Iran.
Cell Mol Biol (Noisy-le-grand). 2024 Sep 8;70(8):76-81. doi: 10.14715/cmb/2024.70.8.9.
Influenza and Newcastle disease are the most important poultry diseases that cause high annual damage to poultry farms worldwide. Newcastle virus fusion (F) gene and Influenza Virus Hemagglutinin (HA) gene are capable of encoding F and HA proteins that are the main factors in creating immunity, so this study aimed to clone and express these genes in Spodoptera frugiperda (Sf9) cells using baculovirus expression system. After isolating the Newcastle and Influenza virus genome, the HA gene of influenza virus and the F gene of Newcastle virus were amplified by reverse transcriptase PCR and specific primers and then cloned into pFastBacTM Dual plasmid. A recombinant sucker with these genes was produced in the DH10Bac host cell. By transfecting Sf9 cells with recombinant bacmid, expression was assessed by SDS-PAGE, western blotting, and Bradford methods. Cloning of genes into the bacmid was successful. By transfecting the recombinant bacmid into Spodoptera frugiperda cells, 218 µg/ml of the recombinant protein was obtained in the supernatant. In addition, the presence of protein was confirmed by western blotting. The PCR products of HA and F genes showed one band of 1.7 kb size using specific primers. The pFastHA1 vector was about 7 kb in size. Two bands of about 7 kb and 1.7 kb were created by ligation of the F gene and pFastHA1 vector based on enzymatic digestion, indicating the correct ligation of F gene under the P10 promoter. This is the first report on the cloning and Co-expression of two HA and F genes using baculovirus expression system and can be a candidate for dual influenza and Newcastle vaccine. Mixtures of these recombinant proteins can be used as vaccine candidates against both avian influenza and Newcastle disease.
流感和新城疫是最重要的家禽疾病，给全球家禽养殖场造成了巨大的年度损失。新城疫病毒融合（F）基因和流感病毒血凝素（HA）基因能够编码 F 和 HA 蛋白，这些蛋白是产生免疫的主要因素，因此本研究旨在使用杆状病毒表达系统在 Spodoptera frugiperda（Sf9）细胞中克隆和表达这些基因。在分离出新城疫和流感病毒基因组后，通过反转录酶 PCR 和特异性引物扩增流感病毒的 HA 基因和新城疫病毒的 F 基因，然后将其克隆到 pFastBacTM Dual 质粒中。在 DH10Bac 宿主细胞中产生了带有这些基因的重组杆状病毒。通过用重组 bacmid 转染 Sf9 细胞，通过 SDS-PAGE、western blot 和 Bradford 方法评估表达。基因成功克隆到 bacmid 中。通过将重组 bacmid 转染到 Spodoptera frugiperda 细胞中，在上清液中获得了 218 µg/ml 的重组蛋白。此外，通过 western blot 证实了蛋白的存在。使用特异性引物，HA 和 F 基因的 PCR 产物显示出 1.7 kb 大小的一条带。pFastHA1 载体的大小约为 7 kb。根据酶切，F 基因和 pFastHA1 载体的连接产生了约 7 kb 和 1.7 kb 的两条带，表明在 P10 启动子下 F 基因的正确连接。这是首次使用杆状病毒表达系统克隆和共表达两种 HA 和 F 基因的报道，可作为双重流感和新城疫疫苗的候选物。这些重组蛋白的混合物可作为针对禽流感和新城疫的候选疫苗。
