적조유발 편모조류 Heterosigma akashiwo에 감염하는 HaRNAV의 바이러스 유사입자 형성

Abstract

In order to control harmful algal bloom that causes red tide, we made an attempt to express and purify viral capsid protein genes from viruses, which isolated from harmful algae. The recombinantly expressed VLPs of HaRNAV (specifically infect and kill the flagellated H. akashiwo) is considered as algaecide. The HaRNAV is T-pseudo3 icosahedronal shape, which consists of three structural capsid proteins such as vp1, vp2 and vp3. The structural gene sequences that encoding HaRNAV capsid proteins were obtained from Genebank (NCBI). These sequences was chemically synthesized with E. coli codon preferences, then cloned into pUC57 vector and then expressed in E. coli BL21 (DE3) host. The VLP genes were cloned into various expression vectors such as pET30a, pET30a-intein, pTXB1, pGEX-4T-1, pMAL-c5x, pET-SUMO and pHCE-IA. These expression vectors posses few drawbacks during cloning, expression, purification and cleavage process. To avoid these problems, the VLPs-TEV protease genes were cloned into pCOLD-TF expression vector, which contains the following specialized genes such as 6xHis (Ni-NTA purification), TF chaperone (increase proper folding and increasing the solubility of the VLPs on N-terminal end) and also other protease genes (Thrombin, FactorXa, HRV3C protease). After cloning, the expression vector also containing TEV protease gene, which helps to cleave the desired VLPs from pCOLD-TF VLPs. The maximum HaRNAV capsid production was obtained at 15℃ in 24h incubation. All the VLPs were purified using Ni-NTA affinity column chromatography. From the SDS-PAGE analysis, it was revealed that the expression systems are capable to produce significant level of VLPs. The molecular weight of TF-vp1, TF-vp2 and TF-vp3 was found to be 82, 72 and 77 kDa, respectively. In addition, TEV protease was produced using recombinant E.coli grown at 37℃ and then 0.3 mM IPTG induction was performed. Then the culture was grown at 16〫℃ for 20h. After incubation, the supernatant was collected and used for TEV protease purification using Ni-NTA column and FPLC. The fractions obtained from FPLC were analyzed using 12% SDS-PAGE and the molecular weight of TEV protease was found to be 27 kDa. Finally, the protease was concentrated to 1 mg/ml and stored at -80℃ until further use. To obtain the desired VLPs, the fusion protein (1 mg/ml) and TEV protease (1mg/ml) optimum ratio for vp1, vp2 and vp3 was found to be 200:1, 50:1 and 10:1, respectively. This mixture was incubated at 30〫℃ for 2 h. After incubation, the products were analyzed using 12 % SDS-PAGE. From the results, it was confirmed that the pCOLD-TF vp1 was not completely cleaved. However, the latter two was completely cleaved under the optimum condition. In order to further purification, a number purification techniques were used including Ni-NTA affinity, size exclusion and ion exchange chromatography. The eluted samples were analyzed using 12% SDS-PAGE. From the results, it was observed that no significant and complete purification of VLP. However, the Ni-NTA affinity chromatographically purified pCold-TF vp1, pCold-TF vp2 and pCold-TF vp3 fusion proteins eluted and dialysed to remove imidazole. All three fusion proteins were mixed to induce self-assembly of vp1, vp2 and vp3 under controlled conditions (fusion protein:TEV protease - 10:1). The formed fusion protein complex was treated with TEV protease at aforementioned conditions. Then the cleaved products were separated by FPLC using sephacryl S-500 packing column. The high molecular weight fractions were collected and analysed using 12% SDS-PAGE. These induced self-assembled VLPs morphology was found to be amorphous structures under TEM analysis. To find out the host specificity binding efficiency of VLPs with host recognition site with the individual vp-FITC and self assembled VPLs-FITC were tested against various host species such as Heterosigma akashiwo (NEPCC 102), Heterosigma akashiwo (NEPCC 522), Chattonella marina (KMMCC579) and Gyrodinium aurelum (KMMCC 978). The host specificity of FITC fluorescence labeled HaRNAV VLPs were analysed under fluorescence microscopy. From the individual vp-FITC host specificity analysis, there is no significant host binding efficiency was found. In addition, the induced and self-assembled VLPs-FITC also not found host specific binding with the aforementioned algal cells.