利用基因重組大腸桿菌表現海鱺淋巴囊腫病毒 之主要鞘蛋白及其單株抗體生產與分析系統之建立

Expression of the Major Capsid Protein of Cobia (Rachycentron canadum) Lymphocystis Disease Virus by recombinant E. coli and the production of its monoclonal antibody

劉楊威(Yang-Wei Liu)

指導教授 ： 陳建源

國立臺灣大學/生命科學院/微生物與生化學研究所/碩士(2005年)

https://doi.org/10.6342/NTU.2005.10110

摘要

本論文以罹患淋巴囊腫病的海鱺為材料，自數種組織及器官分離出基因組DNA (genomic DNA)後，設計專一性引子並進行聚合酶連鎖反應(polymerase chain reaction)，以偵測並放大主要鞘蛋白的DNA序列mcp，經由定序後可知此段序列長度為1295個鹽基(base pair)，利用Bioedit軟體分析其限制切位圖譜(restriction map)以及下游轉譯胺基酸序列組成，推測其下游產物分子量應介於45至50 kD之間。
自含有病毒的器官及組織中分離出病毒顆粒，進行免疫以生產單株抗體，免疫方法包括傳統法以及快速免疫法(RIMMS)，將免疫完成的小鼠脾細胞取出，與骨髓癌細胞NS-1利用PEG 1500進行細胞融合，以HAT(Hypoxanthine、Aminopterin、Thymidine)培養液進行選擇性培養，並以酵素免疫分析法(ELISA)篩選出具分泌抗淋巴囊腫病毒主要鞘蛋白之融合瘤(hybridoma)細胞，共獲得3C6抗體與2A10抗體兩株高效價的單株細胞。分型結果顯示3C6的重鏈為IgG1而2A10的重鏈為IgG2a，兩者的輕鏈均為κ。其中3C6單株細胞生長快速且分泌出的抗體效價較高，以之作為生產抗體細胞株。將其產生的單株抗體進行純化。步驟包括先以硫酸銨沉澱後，以DEAE-Sepharose離子交換樹脂與Protein-G管柱進行親和性層析，再以原態膠體電泳、變性膠體電泳(SDS-PAGE)與ELISA進行分析。
最後將mcp基因以pQE31表現載體於大腸桿菌DH5α中進行蛋白質生產，pQE31上帶有His-tag，因此重組蛋白質可利用Ni-NTA親和管柱進行純化，純化後的MCP蛋白質經由SDS-PAGE分析後可知其分子量約48kD，而經由ELISA與西方墨點染色(Western blotting)而知其可與單株抗體3C6結合。
以單株抗體進行海鱺淋巴囊腫病毒主要鞘蛋白的檢測，利用間接ELISA的方式，將純化後的3C6抗體經稀釋104倍後使其與不同濃度的MCP進行結合並呈色，實驗結果顯示檢測範圍介於0.5至5 μg/mL 時有良好的線性關係，可偵測MCP的極限為0.5 μg/mL。
由以上結果可知，針對淋巴囊腫病毒主要鞘蛋白之單株抗體可應用於進行病毒顆粒的檢測，並可能進一步發展如免疫反應型生物感測器等快速檢測平台。此外，利用基因重組大腸桿菌大量生產主要鞘蛋白MCP，亦具有發展成魚用疫苗的潛力。

關鍵字

海鱺淋巴囊腫病 ； 虹彩病毒 ； 主要鞘蛋白 ； 單株抗體 ； 快速免疫法RIMMS

並列摘要

The material of this study is the Cobia(Rachycentron canadum) which were infected by iridovirus and had lymphocystis syndrome. We isolated the genomic DNA of several organs and tissues and designed specific primers to detect and amplify the major capsid protein gene mcp by polymerase chain reaction (PCR). After sequencing, the total length of mcp is 1295 base pairs. Then, we used the software Bioedit to analyze the restriction map and simulate its translation product, MCP. The number of amino acid composing MCP is 431, which suggested that the molecular weight should be between 45 to 50 kD.
We isolated viral particles from infected organs and tissues by blending method and emulsified viral particles to immune Balb/c female mouse in order to produce monoclonal antibody (mAb). Immunization methods are traditional method and RIMMS. Then the spleen cells of immuned Balb/c mouse were collected and fused with NS-1 myeloma cells by PEG-inducing method. The fused cells were then selected in HAT medium. The hybridomas, which could secrete anti-MCP antibodies, were screened with enzyme-linked immunosorbant assay (ELISA). Then the hybridomas were subcloned by limit dilution. Two hybridomas producing anti-MCP mAb were obtained and designated as 3C6 and 2A10. The isotypes of 3C6 and 2A10 were IgG1 and IgG2a as heavy chain and same κ as light chain. 3C6 hybridoma secreted higher titer of anti-MCP antibodies and had better growth conditions than 2A10 hybridoma, thus we chose 3C6 hybridoma to produce anti-MCP mAb. Then we purified antibodies by following methods: ammonium sulfate participation, ion-exchange (DEAE) chromatography, and Protein-G affinity chromatography. SDS-PAGE and ELISA were used to analyzed purified products.
Finally, we cloned mcp gene into pQE31 expression vectors and expressed MCP by E. coli DH5α. The recombinant MCP protein was purified easily because it carried 6x His-tag on N-terminal. After Ni-NTA affinity chromatography, pure MCP protein was collected, and its molecular weight was about 48 kD. Western blotting showed that recombinant MCP would interact with 3C6 mAb specifically.
With indirect ELISA, purified 3C6 antibodies were diluted to 104 times to interact with different concentrations of MCP protein. The MCP concentration ranged form 0.5 to 5 μg/mL showed a better linear relationship, and the detection limit was 0.5 μg/mL.
In summary, we produced antibodies which interacted with major capsid protein of iridovirus that were isolated from cobia with lymphocystis syndrome. We hope that the mAb produced in this study can apply in virus detection. Otherwise, the recombinant MCP also has a potential to develop as vaccines.

並列關鍵字

Cobia lymphocystis disease ； iridovirus ； major capsid protein ； monoclonal antibody(mAb) ； RIMMS.

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