禽類IgY抗體在新冠病毒之保護、預防與檢測研究

The study of avian IgY antibody in the protection, prevention and detection of novel coronavirus

葉嘉翠(Yeh Chia-Tsui)

指導教授 ： 李冠群

國立臺灣師範大學/理學院/生命科學系/博士(2023年)

https://doi.org/10.6345/NTNU202300310

摘要

因應對生物醫學研究的需求，實驗動物的福祉及符合動物實驗3R的要求日益提升。過去以小鼠、兔子、山羊、牛、馬…等傳統動物模式來產製多株抗體的方式，將被嚴格的監督、要求與限制。所以尋找取代動物抗體產製的新方法及動物模式，成為一個非常重要的課題。蛋黃免疫球蛋白（yolk immunoglobulins，IgY），是卵黃中唯一的免疫球蛋白，以動物種源發生學的距離，哺乳類與鳥類的同源性低的優勢，可以產生具有高專一性的抗體，在某些免疫分析中，常用雞卵黃 IgY 替代哺乳類 IgG。IgY 特殊的分子結構與哺乳類免疫球蛋白略有差異，以至於不會引發有害人體的補體反應，也不會與類風濕性因子（rheumatoid factor）結合，也不會與哺乳動物的 IgG 產生交互作用（cross reaction）。只要收集免疫抗原後的雞蛋，加以純化取得卵黃中的抗體即可，不用犧牲動物或是長期侵入性的抽血取得動物血清。雞卵黃具有非侵入性、可持續高產量、飼育及純化成本低的種種優點，成為本研究中產製多株抗體的首選動物模式。本研究以抗原免疫蛋雞後，待蛋雞血清中專一性抗體濃度升高後，將蛋雞產出的雞蛋進行卵黃 IgY 抗體的純化，並進行抗體的專一性及靈敏度測試。
本研究以新型冠狀病毒（SARS-CoV-2）之刺突蛋白（又稱棘蛋白）（spike protein or S protein）蛋白次單元蛋白 S1、S2 誘發的 IgY 中和抗體，在 Vero E6 細胞株的感染中和力試驗中證明具有良好阻抗病毒的效果，進一步在倉鼠鼻滴定病毒感染試驗中，一次性投予抗 S 蛋白之 IgY 中和抗體，能有效降低新型冠狀病毒感染及病徵的嚴重程度。而 SARS-CoV-2 新型冠狀病毒抗N（nucleocapsid proteins；核殼蛋白）蛋白 IgY 抗體，則可專一且靈敏的於 11 株人類常見的呼吸道病毒試驗的酵素結合免疫吸附分析法（ELISA）試驗中，在 100~1000 個病毒的狀態下，檢測出 SARS-CoV-2 的病毒株。結果顯示誘發的 IgY 抗體具有高抗體效價特、專一性及中和力價。本研究亦誘發抗 SARS-CoV-2 病毒的 IgY 抗體，並且利用 IgY 抗體進行 ELISA、Western Blot、IHC、IF 等試驗的運用。總而言之，本研究證明了 IgY 抗體具有抵抗呼吸道傳染性病原的能力，可發展成具阻抗病原感染的預防和治療的中和性抗體藥品，及發展成更優良的檢測工具。

關鍵字

蛋黃免疫球蛋白 ； 新型冠狀病毒 ； 刺突蛋白或棘蛋白 ； 核殼蛋白或N蛋白

並列摘要

The requirements for animal experiments in biomedical research, the welfare of experimental animals and the compliance with the 3Rs of animal experiments are becoming more and more stringent. In the past, traditional animal models such as mice, rabbits, goats, cows, and horses were used to produce polyclonal antibodies, which will be strictly supervised, required, and restricted. Therefore, finding new methods and animal models to replace traditional animal antibody (Ab) production has become a very important topic. Egg yolk immunoglobulins (yolk immunoglobulins, IgY) are the only immunoglobulins in egg yolk. In terms of evolution, mammals and birds have very low homology. IgY can be used as a highly specific antibody for the application in mammals. In some immune assays chicken egg yolk IgY is often used instead of mammalian IgG. The special molecular structure of IgY is slightly different from that of mammalian immunoglobulins, so that IgY does not trigger harmful human complement responses, nor does it bind to rheumatoid factor and cross react with mammalian IgG. It is only necessary to collect eggs after immunization with antigens and purify antibodies in the yolk without sacrificing animals to obtain animal serum or long-term invasive blood draws. The advantages of non-invasiveness, sustainable high yield, and low cost of breeding and purification have made chicken egg yolk IgY become the primary choice for the development of animal models for producing polyclonal antibodies in this research. In our research, after immunizing laying hens with antigens, the concentration of specific antibodies in the serum of laying hens increased. The egg yolk IgY antibodies were purified from eggs produced by laying hens, and the specificity and sensitivity of the antibodies were tested.
The subunit proteins, S1 and S2, of the spike protein (S protein) of the new coronavirus SARS-CoV-2 which are human respiratory infectious pathogens were used to induce IgY neutralizing antibodies. In the infection neutralization test of the Vero E6 cell line, it has been proved that the IgY has a good effect of resisting the virus infection. Further, in the hamster nasal titration virus infection test, one-time administration of IgY neutralizing Ab against the S protein can effectively reduce the infection of the new coronavirus and severity of symptoms. In the test of 11 strains of common human respiratory viruses by enzyme-linked immunosorbent assay (ELISA) test, the IgY Ab against N (nucleocapsid) protein of SARS-CoV-2 specifically and sensitively detected the strain of SARS-CoV-2. The results suggest that the induced IgY antibody shows high antibody titer, specificity, and excellent neutralizing potency. In this research, SARS-CoV-2 virus was also used to induce IgY antibody, and the ELISA, Western Blot, IHC, IF and other tests using the induced IgY antibody were performed. In conclusion, it has been confirmed that IgY antibodies were able to resist respiratory infectious pathogens, and they could be applied to develop neutralizing antibodies drugs for resisting pathogenic infection, and develop a better detection tool in the future.

並列關鍵字

yolk immunoglobulins (IgY) ； SARS-CoV-2 ； coronavirus ； spike protein (S protein) ； nucleocapsid protein (N protein)

參考文獻

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