1308929 九、發明說明： ^ 【發明所屬之技術領域】 本發明係關於一種篩選高產蛋禽類之方法，特別是關 於一種篩選高產蛋土雞之方法。 【先前技術】 土雞的飼養係藉由土雞在開放環境自由行動與運動而 Φ 提升其肉質，由於土雞的肉質細嫩而大受部分亞洲國家與 歐洲國家的喜愛，特別在華人社會中常用來準備多種滋養 食補。然而，土雞大致上較商業產蛋雞具有較低的產蛋性 能（李淵百，1992。台灣的土雞。國立中興大學畜牧學 ' 系，台中，台灣。56頁）。 - 通常土雞具有較複雜且較不整齊的基因組合（戴謙、 鍾秀枝、張秀鑾、黃祥吉。1997。台灣土雞之近親育 種：VI.近親品系二元雜交後裔之生長及繁殖性能隻田間評 φ 估。中畜會誌26(2): 187-196)，在大部分的家禽養殖農場， 不論是小規模或商業規模的農場，土雞都並非以系統化的 交互配種（cross-mating)來保存其品種，而常是從雞群中選 擇肉質較佳的土雞作為培育的種雞。然而這些種雞一般較 其他產蛋雞具有較低的產蛋性能，有些種雞甚至一個產期 僅產下少於70個蛋（鍾秀枝，1998。台灣省畜產試驗所四 十週年慶家畜禽遺傳育種研討會論文集PP.121-132)。當土 雞的全球市場目前仍持續增加，卻苦於無法增加土雞的產 量以維持市場需求。 1308929 【發明内容】 心本發糊W產蛋禽類的 科泛本毛月#面提供一種篩選高產蛋禽類的方法，其特 作)fc 類姨島素生長因子 1 (insulin 他e growth factor 1 (IGF;) 測財法包括㈣職禽賴檢體，在檢體内檢 、、素生長因子1的總量，其中高產蛋禽類的判斷是 根據檢體内所檢測的類胰島素生長因子ί含量。 本發明另—方面亦提供賴島素生長因子 基：9包括至少序列辨識號1卿卿:”的胺基酸ί 胰島方面亦提供一抗體’其可專-性結合類 二長□子I的—抗原決定區’包括至少 DN〇 1的胺基酸殘基75至89。 . 本發明又-方面提供一種篩選高產蛋禽類 ，徵為以類胰島素生長因子】作為指標中產黍金 =決定於檢體内所檢測的類联島素生長二= 素生長因子百 五。又根據本發明之另_具„施例分之十 群中類騰島素生長因子I含量較高之百分之^擇又測禽 如百分之二。 ^一至二十’例 本發明再-方面提供一種筛選高產 方法包括收集測試禽類的檢體，以一抗 員：方法’該 /、j專一性結合 1308929 類胰島素生長因子1的一抗原決定區，包括至少SEQID • ΝΟ:1的胺基酸殘基75至89，檢測檢體内類胰島素生長因 ‘子的總量’其中高產蛋禽類的判斷是根據檢體内所檢測的 類胰島素生長因子1是否含相較高量。 【實施方式】 為使本發明内容清楚易懂，以下將對所使用的專有名 詞做更詳盡的解釋。 在此所使用的冠詞“一”意指一或多於一（也就是至 • 少一）的文法物件之冠詞，除非是另行解釋該冠詞僅為以 單一觀念的特定使用。 “抗原決定區”在此係指蛋白質上的單一抗原位置與 抗體反應。抗原一般具有數個不同抗原決定區，並與抗體 具有不同專一性的反應。 “免疫體” 一詞所指的係任何當進入體内而引起或產 生免疫反應的物質或生命體。 本發明提供一種篩選高產蛋禽類的方法，其特徵為以 & 類胰島素生長因子 I (insulin like growth factor I (IGF-I))作為指 標。 根據本發明一實施例，係提供類胰島素生長因子I的 抗原決定區’包括至少SEQ ID NO: 1的胺基酸殘基75至 89。該抗原決定區係位於類胰島素生長因子I中間的高親 水性、易彎曲性以及表面化區域。 該抗原決定區可合成為一胜肽，該抗原決定區也可與 其他分子連接或結合以形成蛋白集合體❻⑺访出 1308929 aggregates)、抗原蛋白或免疫體，在進入宿主動物體内可 促進免疫反應以產生相對抗體。抗原決定區的判斷可以藉 . 助生物資訊軟體尋找一大群參與產蛋研究相關之生物路徑 的蛋白質指標。 本發明也提供一抗體，其可專一性結合類胰島素生長 因子I的一抗原決定區，該抗原決定區包括至少SEQID NO: 1的胺基酸殘基75至89，其中該抗體可為多株抗體 (polyclonal antibody)或單株抗體（monoclonal antibody)。 • 本發明也提供一種篩選高產蛋禽類的方法，其中該高 產蛋禽類係決定於檢體内所檢測的類胰島素生長因子I含 量。根據本發明之一具體實施例，其中係淘汰受測禽群t 類胰島素生長因子I含量較低之百分之五至五十，例如百 分之十五。又根據本發明之另一具體實施例，其中係選擇 ' 受測禽群中類胰島素生長因子I含量較高之百分之二至二 十，例如百分之二。 根據本發明之一具體實施例，該方法包括由測試禽類 • 收集檢體，以專一性結合類胰島素生長因子I的抗原決定 區的抗體，檢測檢體内姨島素生長因子I的含$ ’該抗原 決定區包括至少SEQ ID NO: 1之胺基酸殘基75至89，其中 高產蛋禽類的判斷是根據檢體内所檢測的蛋白質含量是否 含相較高或低量。在此相較高量的蛋白質係定義為以專一 性結合類胰島素生長因子I的一抗原決定區，包括至少 SEQ ID NO: 1之胺基酸殘基75至89的抗體，在禽群中選擇 所檢測蛋白質含量較高之禽類，或淘汰所檢測蛋白質含量 1308929 較低之禽類。例如，當農場主人欲選擇此禽群中百分之五 之高產蛋禽作為種禽，他將在禽群中選擇血清檢體檢測出 . 類胰島素生長因子I含量較高的百分之五作為種禽。又 如，當農場主人欲淘汰此禽群中百分之十五低產蛋禽，他 將在禽群中選擇血清檢體檢測出類胰島素生長因子I含量 較低的百分之十五作為淘汰禽類。 本發明所使用的檢體可包括測試禽類的細胞、組織或 體液。在一實施例中，檢體包括由測試禽類所收集的血 Φ 清，該血清可根據習知血液學技術由測試禽類所抽取的全 血所製備。 根據本發明，測試禽類包括雞類、鴨類、鵝類、鴿 類、有色鳥類以及罕見鳥類。根據特定實施例，該雞類為 ' 土雞類包括但不限於商用紅羽土雞、來亨蛋雞以及台灣紅 羽土雞。 在一較佳實施例，進行西方轉潰分析以辨識台灣紅羽 善 土雞血清内的類胰島素生長因子I。如圖1所示，該抗體 在高產蛋群組之台灣紅羽土雞血清内辨識出具有一前驅物 大約13.7kDa以及一成熟蛋白質大約7.0kDa的類胰島素生 長因子I。然而，也可進行其他免疫分析方法，包括具有 抗體固定於其中的酵素免疫吸附檢測（enzyme-linked immunosorbent assay (ELISA))以及蛋白質晶片，以決定血清 内類胰島素生長因子I的含量。 根據另一實施例，本發明抗原決定區的胜肽在使宿主 10 1308929 動物產生免ίΐ應前’可進—步以多重抗原胜肽修改技術 進订修：：：攸宿主動物收集所產生的抗體。 * 產吐Γ例中’抗體是藉由免疫體組成使宿主動 —抗體的兔子產生免疫反應所製備。本發明 產生抗體=主動，包括但不限於牛、馬、驢、山羊、綿 1、tut以及其他市售用於產生抗體的實驗動 ^ ’只要“動物本身並未料切抗原蚊區的胜肽 序列。 •^生的方法為熟知該項技藝者所知並包括皮 熟知此項技藝者可根據將產生免疫 的！抗原性以及注射位置改變產生免疫 反應所使㈣纽體量。增加抗祕㈣法㈣知剛項技 藝者所知且包括但不限於在祕性蛋自(heter〇i〇g⑽ _in)，如球蛋白或beta半乳糖酵素（P-galactosidase)連接 抗原’或透過在產生免航料包含佐藥㈣_〇。 抗體根據本發明另-實施例，該抗體可為多株抗體或單株 關於單株抗體的取得，將產生免疫反應之宿主動物的· ，臟細胞移除，與骨髓癌細胞融合（fUse)，使其變成產生. 單株抗體的融合瘤細胞（hybridoma eells)。 可以任何熟知該項技藝者所知的數個方法之一辨識出 產生具有所需特徵之抗體的融合瘤細胞，這些包括以點潰 分析（dotblotanalysis)、酵素免疫吸附檢測、西方轉潰分 析或反應性免疫分析。複製分泌所需抗體的融合瘤細胞， 1308929 並且根據習知步驟判斷免疫球蛋白種類與次種類。 關於多株抗體的取得’從產生免疫反應的動物分離包 含抗jk清的抗體’並以上述步驟之—篩選具有特定專一性 之抗體的存在。 上述的抗體係以可彳貞測的標定形式使用，抗體可以透 過使用放射性同位素(radioisotope)、親和性標定物（如生物 素、抗生物素蛋白等）、酵素標定物（如辣根過氧化酶 (horseradish peroxidase )或鹼性磷酸酶(alkalineph〇sphatase) 鲁 專）、螢光疋物（如螢光黃或若丹明（rhodamine)等）、 順磁原子（paramagnetic atoms)等。完成標定的步驟為熟知該 項技藝者所知’本發明所標定的抗體可用於體外、體内以 及原位分析(in situ assay)以便在禽類的血清或檢體液内辨識 類胰島素生長因子I (或其片段），較佳的免疫分析為習 知的不同種類酵素免疫吸附檢測以及反應性免疫分析。抗 體本身可直接用於治療或其他的診斷方法。 上述抗體可以固定在固體支撐物，固體支撐物的範例 _ 包括塑膠，如聚礙酸酯（polycarbonate)、複合碳水化合物 (complex carbohydrates)如瓊脂糖（agarose)以及瓊脂糖凝膠 (sepharose)丙稀酸樹脂(acrylicresin)以及如聚丙稀醯胺 (polyacrylamide)以及乳膠珠（latex beads)。將抗體連接在固 體支撐物上的技術為熟知該項技藝者所知，所固定的抗體 可用於體外、體内以及原位分析(in situ assay)並且可用於在 禽類的血清内辨識類胰島素生長因子I。因此可將抗體固 定或結合在蛋白質晶片或可攜式感應裝置以檢測測試禽類 12 1308929 之檢體内_胰島素生長因子i，以便根據檢體内所檢測 的蛋白質含量篩選高產蛋禽類。 本發明進一步以下列實例予以闡明。下列實例係屬說 明性質，無意限制本發明之範疇。 實例一搜尋高產蛋土雞之蛋白指標 本實驗使用35週的台灣紅羽土雞，根據台灣紅羽土雞 於26至48週產蛋總數分為高產蛋群組與低產蛋群組，如表 一所不’高產蛋群組的雞隻在26至48週的平均產蛋數大約 為117個’而在26至48週的平均產蛋數大約為69個的雞隻 則被列為低產蛋群族。 表一 -^ __ 組別 樣品數 產蛋數（個） 高產蛋組 19 117±1* 低產蛋組 18 69±4* 差異顯著程度p<〇.〇5 爲找出與產蛋相關的蛋白指標，從每一組台灣紅羽土 雞抽取青春期前(pre_pubertal)階段（第14週）與成熟期 (mature)階段（第35週）血液檢體，血液離心後取上清 液，定篁、乾燥後加入溶解液（lysis buffer)包含： 7摩爾（Μ)尿素（urea)、2摩爾硫尿素（thiourea)、 4%二甲基氨丙橫酸 (3 - [(3 -cholamidopropyl)dimethylammonio] -1 -propane sulphonate, CHAPS)、2% 二硫蘇糖醇（Dithiothreitol，DTT)以及 2% pH 4-7 固相化 pH 梯度緩衝液（imm〇biliZed pH gradient buffer, 13 1308929 IPG buffer)以溶出蛋白質。 於4°C以10,000離心力值（g)離心15分鐘後，取出 400微克的上清液，以供進行雙向蛋白質電泳分析。 雙向蛋白質電泳分析工作分成第一維之等電聚焦電泳 (isoelectric focusing，IEF)和第二維之SDS聚丙烯醯胺膠體電 泳分析（SDS-polyacrylamide gel electrophoresis，SDS-PAGE)。使 用固相化pH梯度，以載體二性電解質附著丙烯醯胺分子 並鑄成膠體以形成固定的pH梯度，使用iPGphor系統 (Amersham Biosciences)對蛋白質進行1〇〇,〇〇〇伏特小時 (voltage-hour，VH)的電場’之後以SDS平衡緩衝液（SDS equilibration buffer)包含50亳摩爾三（羥甲基）氨基曱烷鹽 酸鹽（Tris-HCl) pH8.8、6 摩爾尿素、30% 甘油（glycerol)以 及0.02%SDS水和膠條，再將平衡的ipg膠條放入1〇_15% 梯度SDS電泳膠上緣，使用Daltsix直立式電泳系統 (Amersham Biosciences)以含有 SDS 緩衝液的 1 % agarose 包埋 起來’進行第二維的電泳分析，使用的電壓為200伏特 (V) ’電流為30毫安培(mA) ’直到染劑行進至玻璃片下緣 時終止電泳工作。 將第二維電泳分析完成的膠片浸泡於固定溶液 (fixation solution)包含10%醋酸與40%甲醇10分鐘，再浸泡 於水和溶液(rehydration solution)中至少5分鐘，之後用二次 蒸餾水（二次水）清洗3次。將膠片浸泡在含一滴溶液A (SolutionA; 80毫克/毫升硫酸鈉（Na2S〇4))的1〇〇毫升二次 水中10分鐘，以二次水清洗2次，加入含1毫升溶液b 1308929 (Solution B; 100毫克/毫升硝酸銀（AgN03))的50毫升二次水 作用10分鐘，以二次水清洗1次，加入含40毫升二次水、 10毫升溶液C(SolmionC;150亳克/毫升碳酸鈉（Na2C03))、 和一滴溶液D (Solution D; 37.5% 曱藤(formaldehyde))的溶液 5〇 毫升作用10秒後倒掉，再加入相同的溶液呈色，直至膠體 以石肖酸銀顯色完全後，加入2毫升醋酸（acetic acid)中和反 應，之後將膠體移至二次水中，保存於4°C。 將經過雙向蛋白質電泳及銀染色過後之銀染膠片上目 φ 標蛋白質點一一切下’浸泡於退染緩衝液(destaining buffer) (15 毫摩爾亞鐵氰化鉀（p〇tassium ferriCyanide [K3Fe(CN)6])， 5〇毫摩爾硫代硫酸鈉（sodium thiosulfate [Na2S2〇3]))中直到膠 片完全褪色。將膠片置於1毫升25毫摩爾碳酸氫銨 (NH4HC〇3)中輕微震盪1〇分鐘，去除上清液，再置入i毫 升25毫摩爾碳酸氫銨/5〇%乙腈（acet〇nitriie)中輕微震盪1〇分 鐘’去除上清液’此時膠體稍微皺縮，再利用真空乾燥機 完全脫乾。加入50毫升25毫摩爾碳酸氫銨和1毫升β-毓基 籲 乙醇（P-mercaptoethanol)避光下作用20分鐘，再加入50微升 25毫摩爾碳酸氫銨/5〇〇/0乙腈及5微升冬乙烯基吡啶 (4_vinylpyridine) ’避光下作用2〇分鐘，去除上清液，再重 複上述步驟中將膠片置於5〇微升25毫摩爾碳酸氫銨和5〇奈 克修飾胰蛋白（modified trypsin) (Promega)於 37°C 作用 18 小 時’收集上清液’並用200微升0.1〇/〇甲酸（formicacicl)清 洗膠片’輕輕混合20分鐘，收集上清液並與之前的上清液 混合’以真空乾燥機乾燥後保存在_2〇乞，等待液相層析 15 1308929 串聯質譜儀(LC/MS/MS)分析，液相層析串聯質譜儀結合了 高壓液相層析儀(HPLC)的溶質分離力以及質譜儀敏銳的偵 測能力，高壓液相層析儀係根據數個特定胜肽的種專一特 性，如電荷、大小、疏水性以及存在的特定標籤（tag)或胺 基酸分離胜肽。 實例二抗體合成 使用實例一所預測的抗原決定區為源於序列辨識號1 (SEQ ID NO: 1)之胺基酸殘基75至89的15個胺基酸胜肽。此 胜肽具有一序列KPTGYGSSSRRLHHK位於類胰島素生長因 子I中間的高親水性、易彎曲性以及表面化區域，且較佳 具有二級蛋白質結構，如a螺旋（α-helix)以及b平面 (β-sheet)。 胜肽的合成是以多重抗原胜肽（multiple antigenic peptide) 型式合成。此種胜肽分子量大約為8〜18 kDa，多重抗原胜 肽的中心區域為包含7個離胺酸（lysine)鍵結8個胜肽鏈往 外延伸，在抗體的製造上，不需和攜帶蛋白質的結合便可 獨立當作免疫抗原。所有合成的胜肽須需經由高壓液相層 析儀及質譜儀的分析加以確認其品質。接著將胜肽注射到 兔子以產生多株抗體專一性連接胜肽包括序列辨識號1之 胺基酸殘基75至89的抗原決定區。從產生免疫反應的兔子 内分離含抗血清的抗體並使用習知點潰分析篩選具有特定 專一性的抗體。 藉由西方轉潰分析法分析抗體以辨識台灣紅羽土雞血 16 1308929 清内的類胰島素生長因子I，透過上述SDS聚丙烯醯胺膠 體電泳分析分離血清内的蛋白質後，將膠體上的蛋白質轉 移到硝化纖維（nitrocellulose)或聚二氟乙稀(PVDF)膜，在阻 斷液（blocking solution; 100毫升清洗緩衝液（2〇毫摩爾三 (羥甲基）氨基甲烷鹽酸鹽pH7.4、500毫摩爾氯化鈉、 0.05%吐溫20(Tween-20))以及3%明膠）内將上述膜阻斷 一小時’再以清洗緩衝液清洗。加入以清洗緩衝液及1% 明膠稀釋的上述多株抗體與膜產生結合，接著加入鹼性磷 解酶接合抗兔子免疫球蛋白G(alkaline ph〇sphatase e()njugated anti-rabbiUgG)的二級抗體以結合多株抗體，其結合抗原決 定區包括至少序列简號丨的㈣酸 移到鹼性磷解酶顯影液以進杆 打朕将 雷射光密度儀掃描以量化蛋* 風乾顯影後的膜並以 體在高產蛋群組之台灣紅羽土魏含1°請參照圖1 ’該抗 物大約i3.7kDa以及一成雉血清内辨識出具有-前驅 生長因子ι(電轉體之第丨、t自f A^7.GkDa的類騰島素1308929 IX. Description of the invention: ^ [Technical field to which the invention pertains] The present invention relates to a method for screening high-yielding eggs and poultry, and more particularly to a method for screening high-yield egg-laying chickens. [Prior Art] The breeding of chickens is based on the freedom of movement and movement of the chickens in the open environment. Φ Improves the meat quality. Because of the delicate meat quality of the chickens, it is greatly loved by some Asian countries and European countries, especially in Chinese society. To prepare a variety of nourishing food supplements. However, native chickens generally have lower egg production performance than commercial laying hens (Li Yuanbai, 1992. Taiwanese chickens. National Chung Hsing University Animal Husbandry, Department, Taichung, Taiwan. 56 pages). - Usually the chickens have a more complex and less tidy genetic combination (Dai Qian, Zhong Xiuzhi, Zhang Xiuzhen, Huang Xiangji. 1997. Taiwanese close relatives breeding: VI. The growth and reproductive performance of the offspring of the two-bred hybrids only field evaluation φ Estimated. Zhongyuhuizhi 26(2): 187-196), in most poultry farms, whether small-scale or commercial-scale farms, the chickens are not systematically cross-mating. The variety is preserved, and the chicken with better meat quality is often selected from the flock as a breeding breeder. However, these breeders generally have lower egg-producing performance than other laying hens, and some breeders produce less than 70 eggs at a time of birth (Zhong Xiuzhi, 1998. Taiwanese Animal Production Laboratory forty-year anniversary livestock and poultry Proceedings of the Genetic Breeding Symposium PP.121-132). When the global market for chickens continues to increase, it is unable to increase the production of chickens to maintain market demand. 1308929 [Summary of the Invention] The heart of the paste of the eggs and poultry of the family of the Pan Maoyue # face provides a method for screening high-yielding eggs and poultry, special for the fc-type 姨 素 素 生长 growth factor 1 (insulin he e growth factor 1 (IGF;) The method of measuring wealth includes (4) the examination of the body of the poultry, the total amount of the growth factor 1 in the examination, and the judgment of the high-yielding egg and poultry is based on the insulin-like growth factor ί detected in the body. In addition, the present invention also provides a lysin growth factor group: 9 includes at least a sequence identification number 1 qingqing: "the amino acid ί islet also provides an antibody" which can be specifically combined with a long scorpion The -antigenic region of I includes at least DN〇1 amino acid residues 75 to 89. The present invention further provides a screening for high-yielding egg-like birds, which is characterized by the use of insulin-like growth factors as an indicator of the production of gold-free gold. It is determined that the genus-like growth factor detected by the test body is one hundred and five growth factors. According to the invention, the percentage of the growth factor I in the group of ten groups is higher. The choice of the bird and the measurement of the bird is 2%. ^ One to twenty' cases of the invention - Providing a screening high-yield method comprising collecting a test poultry specimen to an antibody: the method 'this/, j-specifically binds to an epitope of 1308929 insulin-like growth factor 1, including at least SEQ ID: ΝΟ:1 Amino acid residues 75 to 89 are used to detect the growth of insulin-like factors in the body. The high-yielding egg-like birds are judged based on whether the insulin-like growth factor 1 detected in the body contains a relatively high amount. [Embodiment] In order to make the content of the present invention clear and easy to understand, the following terms will be explained in more detail. The article "a" as used herein means one or more than one (ie, to 1) The grammar of the article, unless otherwise explained, the article is only for the specific use of a single concept. "Antigenic region" refers herein to a single antigenic position on a protein that reacts with an antibody. The antigen generally has several different antigenic decisions. Zone, and has a specific response to antibodies. The term "immune body" refers to any substance or organism that causes or produces an immune response when it enters the body. Provided is a method for screening high-yielding egg poultry, characterized by & insulin like growth factor I (IGF-I) as an index. According to an embodiment of the present invention, an insulin-like growth factor I is provided. The epitope region comprises at least amino acid residues 75 to 89 of SEQ ID NO: 1. The epitope is located in the high hydrophilicity, flexibility, and surface area of insulin-like growth factor I. It can be synthesized as a peptide, and the epitope can also be linked or combined with other molecules to form a protein aggregate (7) to access 1308929 aggregates, antigenic proteins or immune bodies, which can promote immune response to enter the host animal to produce relative antibody. The determination of the epitope can be borrowed. The bioinformatics software finds a large group of protein indicators involved in the biological pathways associated with egg production research. The present invention also provides an antibody which specifically binds to an epitope of insulin-like growth factor I, the epitope comprising at least amino acid residues 75 to 89 of SEQ ID NO: 1, wherein the antibody may be multiple A polyclonal antibody or a monoclonal antibody. • The present invention also provides a method of screening high-yielding egg poultry, wherein the high-yielding egg-and-bird is determined by the amount of insulin-like growth factor I detected in the specimen. According to a specific embodiment of the present invention, the content of the insulin-like growth factor I in the test group is eliminated by 5 to 50%, for example, 15%. According to still another embodiment of the present invention, wherein the content of the insulin-like growth factor I in the test flock is selected to be from two to twenty percent, such as two percent. According to a specific embodiment of the present invention, the method comprises: collecting a sample from a test subject, and specifically binding an antibody to an epitope of insulin-like growth factor I, and detecting the growth factor I of the insulin-containing factor I in the body. The epitope comprises at least amino acid residues 75 to 89 of SEQ ID NO: 1, wherein the high-yielding egg-like birds are judged whether the protein content detected in the sample contains a higher or a lower amount. Here, a relatively high amount of protein is defined as an epitope that specifically binds to insulin-like growth factor I, including at least an antibody of amino acid residues 75 to 89 of SEQ ID NO: 1, selected among flocks. Poultry with higher protein content detected, or eliminated birds with a lower protein content of 1308929. For example, when a farm owner wants to select five percent of the high-yielding egg poultry in the flock as a breeder, he will select a serum sample from the flock to detect a higher percentage of insulin-like growth factor I. Breeding birds. In another example, when the farm owner wants to eliminate 15% of the low-yielding eggs in the flock, he will select a serum sample from the flock to detect a 15% lower level of insulin-like growth factor I as a phase-out. birds. The specimen used in the present invention may include cells, tissues or body fluids for testing birds. In one embodiment, the specimen comprises blood Φ collected by the test birds, which serum can be prepared from whole blood drawn from the test birds according to conventional hematology techniques. According to the present invention, poultry are tested including chickens, ducks, geese, pigeons, colored birds, and rare birds. According to a particular embodiment, the chicken is 'community chickens including, but not limited to, commercial red-feathered chickens, Lai Hens, and Taiwanese red-feathered chickens. In a preferred embodiment, a western collapse analysis is performed to identify insulin-like growth factor I in the serum of Taiwan Red Feather Chicken. As shown in Fig. 1, the antibody recognized an insulin-like growth factor I having a precursor of about 13.7 kDa and a mature protein of about 7.0 kDa in the serum of Taiwan red feather chicken of the high-yielding egg group. However, other immunoassay methods can be performed, including an enzyme-linked immunosorbent assay (ELISA) and a protein wafer to which the antibody is immobilized to determine the level of insulin-like growth factor I in the serum. According to another embodiment, the peptide of the epitope of the present invention can be further modified by the multi-antigen peptide modification technique before the host 10 1308929 animal is produced free of charge::: antibody. * In the case of vomiting, the antibody is prepared by immunologically reacting a rabbit with a host-antibody to produce an immune response. The present invention produces antibodies = active, including but not limited to cattle, horses, donkeys, goats, cotton 1, tut, and other commercially available assays for producing antibodies, as long as the animal itself does not cut the peptide of the antigenic mosquito Sequences • The method of birth is known to those skilled in the art and includes those skilled in the art. The person skilled in the art can generate immune responses based on the antigenicity and the position of the injection to produce an immune response (4) the amount of the body. Increase the anti-mystery (4) The method (4) is known to those skilled in the art and includes, but is not limited to, in the secret egg (heter〇i〇g(10) _in), such as globulin or beta-galactosidase (P-galactosidase) linked to the antigen' or through the generation of air-free According to another embodiment of the present invention, the antibody may be obtained by a plurality of antibodies or a single plant, and the host animal of the immune reaction is removed, and the viscera is removed. Bone marrow cancer cells are fused (fUse) to become hybridoma eells that produce monoclonal antibodies. Identification of antibodies that produce the desired characteristics can be performed by any of several methods known to those skilled in the art. Tumor cells, including dotblotanalysis, enzyme immunosorbent assay, Western knock assay or reactive immunoassay. Replication of fusion tumor cells secreting the desired antibody, 1308929 and determination of immunoglobulin species according to conventional procedures With regard to the acquisition of multiple antibodies, the antibody containing the anti-jk clear is isolated from the animal that produces the immune response and the above-mentioned steps are used to screen for the presence of antibodies with specific specificity. The above-mentioned anti-system can be speculated For calibration purposes, antibodies can be used by radioisotopes, affinity assays (eg biotin, avidin, etc.), enzyme assays (such as horseradish peroxidase or alkaline phosphatase). (alkalineph〇sphatase), fluorescein (such as fluorescent yellow or rhodamine, etc.), paramagnetic atoms, etc. The steps to complete the calibration are known to the skilled person. The antibodies labeled by the invention can be used in vitro, in vivo, and in situ assays for sera or detection in birds. The insulin-like growth factor I (or a fragment thereof) is identified in the body fluid, and the preferred immunoassay is a conventional immunosorbent assay for different types of enzymes and a reactive immunoassay. The antibody itself can be directly used for treatment or other diagnostic methods. Can be fixed to solid supports, examples of solid supports _ including plastics, such as polycarbonate, complex carbohydrates such as agarose and sepharose acrylic resin (acrylicresin) and such as polyacrylamide and latex beads. Techniques for attaching antibodies to solid supports are well known to those skilled in the art, and the immobilized antibodies can be used in vitro, in vivo, and in situ assays and can be used to identify insulin-like growth in the serum of birds. Factor I. Thus, the antibody can be immobilized or bound to a protein wafer or a portable sensing device to detect the in vivo insulin-inducing factor i of the test poultry 12 1308929 to screen high-yielding egg birds based on the protein content detected in the sample. The invention is further illustrated by the following examples. The following examples are illustrative in nature and are not intended to limit the scope of the invention. Example 1 Search for protein index of high-yield egg-earth chickens This experiment uses 35-week Taiwanese red-feathered chickens, according to Taiwan's red-feathered chickens, which are divided into high-yielding egg groups and low-yielding egg groups in 26 to 48 weeks. Table 1 shows that the average number of eggs laid in the high-yielding egg group is about 117 in 26 to 48 weeks, and the average number of eggs in the 26 to 48 weeks is about 69. Low-yielding egg group. Table 1 -^ __ Group number of eggs produced (number) High-yielding egg group 19 117±1* Low-yielding egg group 18 69±4* Difference significant degree p<〇.〇5 To find out the egg-related Protein index, from each group of Taiwan red feather chickens, pre-puberty (pre_pubertal) stage (week 14) and maturity (mature) stage (35th week) blood samples, blood supernatant, supernatant, fixed, The lysis buffer after drying contains: 7 moles of urea (urea), 2 moles of thiourea, 4% of dimethylammonium hydride (3 - [(3 -cholamidopropyl) dimethylammonio] - 1 -propane sulphonate, CHAPS), 2% dithiothreitol (DTT) and 2% pH 4-7 pH gradient buffer (13 1308929 IPG buffer) to elute protein . After centrifugation at 10,000 centrifugation (g) for 15 minutes at 4 ° C, 400 μg of the supernatant was taken for two-dimensional protein electrophoresis analysis. Two-dimensional protein electrophoresis analysis was divided into first-dimensional isoelectric focusing (IEF) and second-dimensional SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Using a solid phase pH gradient, the propylene amide molecules were attached to the carrier dimer electrolyte and cast into a colloid to form a fixed pH gradient, and the protein was subjected to 1 Torr using an iPGphor system (Amersham Biosciences). The electric field of hour, VH) is followed by SDS equilibration buffer containing 50 moles of tris(hydroxymethyl)aminodecane hydrochloride (Tris-HCl) pH 8.8, 6 moles of urea, 30% glycerol (glycerol) and 0.02% SDS water and strips, then equilibrated ipg strips onto the upper edge of a 1〇_15% gradient SDS gel, using a Daltsix vertical electrophoresis system (Amersham Biosciences) with SDS buffer 1 % agarose is embedded 'for second-dimensional electrophoresis analysis using a voltage of 200 volts (V) 'current is 30 milliamperes (mA)' until the dye travels to the lower edge of the glass sheet to terminate electrophoresis. The second dimensional electrophoresis analysis of the film is immersed in a fixing solution containing 10% acetic acid and 40% methanol for 10 minutes, and then immersed in water and rehydration solution for at least 5 minutes, followed by secondary distilled water (two Secondary water) 3 times. Soak the film in 1 ml of secondary water containing one drop of Solution A (Solution A; 80 mg/ml sodium sulfate (Na2S〇4)) for 10 minutes, wash twice with secondary water, and add 1 ml of solution b 1308929 ( Solution B; 100 mg/ml silver nitrate (AgN03) in 50 ml of secondary water for 10 minutes, wash once with double water, add 40 ml of secondary water, 10 ml of solution C (Solmion C; 150 g/ml Sodium carbonate (Na2C03)), and a drop of solution D (Solution D; 37.5% saponin) solution 5 〇 ml for 10 seconds, then pour off, and then add the same solution color until the colloid is silver tartaric acid After the color development was completed, 2 ml of acetic acid was added to neutralize the reaction, and then the colloid was transferred to secondary water and stored at 4 °C. After two-way protein electrophoresis and silver staining, the silver-stained film is immersed in a destaining buffer (15 mmol of potassium ferrocyanide) (p〇tassium ferriCyanide [K3Fe] (CN) 6]), 5 mM sodium thiosulfate (Na2S2 〇 3)) until the film is completely fading. The film was gently shaken in 1 ml of 25 mmol of ammonium bicarbonate (NH 4 HC 3 ) for 1 minute, the supernatant was removed, and then 25 ml of ammonium bicarbonate / 5 % acetonitrile (acet 〇 nitriie) was placed. Slightly oscillate for 1 ' 'removal of the supernatant' at this time, the colloid is slightly shrunk, and then completely dried using a vacuum dryer. Add 50 ml of 25 mM ammonium bicarbonate and 1 ml of β-mercaptoethanol in the dark for 20 minutes, then add 50 μl of 25 mM ammonium bicarbonate/5 〇〇/0 acetonitrile and 5 Microliter of winter vinyl pyridine (4_vinylpyridine) 'protected for 2 minutes in the dark, remove the supernatant, and repeat the above steps to place the film in 5 〇 microliters of 25 mM ammonium bicarbonate and 5 〇Nike modified trypsin (modified trypsin) (Promega) 18 hours 'Collect Supernatant' at 37 ° C and clean the film with 200 μl of 0.1 〇 / 〇 formic acid (formicacicl) 'Gently mix for 20 minutes, collect the supernatant and before with the upper The clear liquid mixture was dried in a vacuum dryer and stored in _2 〇乞, waiting for liquid chromatography 15 1308929 tandem mass spectrometer (LC/MS/MS) analysis, liquid chromatography tandem mass spectrometer combined with high pressure liquid chromatography The solute separation force of the instrument (HPLC) and the sharp detection ability of the mass spectrometer. The high-pressure liquid chromatography is based on the specific characteristics of several specific peptides, such as charge, size, hydrophobicity and the presence of specific tags. Or an amino acid to separate the peptide. Example 2 Antibody Synthesis The epitope determined by using Example 1 was 15 amino acid peptides derived from amino acid residues 75 to 89 of SEQ ID NO: 1 (SEQ ID NO: 1). The peptide has a sequence of KPTGYGSSSRRLHHK located in the middle of the insulin-like growth factor I with high hydrophilicity, flexibility and surface area, and preferably has a secondary protein structure, such as a-helix (α-helix) and b-plane (β-sheet ). The synthesis of the peptide is synthesized by a multiple antigenic peptide. The molecular weight of the peptide is about 8~18 kDa, and the central region of the multiple antigen peptide is extended by 8 peptide chains containing 7 lysine linkages, and the protein is not required to be carried in the production of the antibody. The combination can be used as an immune antigen independently. All synthetic peptides must be analyzed for their quality by analysis with a high pressure liquid phase analyzer and mass spectrometer. The peptide is then injected into the rabbit to produce a multi-drug antibody-specific linker comprising the epitope of amino acid residues 75 to 89 of SEQ ID NO: 1. Antiserum-containing antibodies were isolated from rabbits that produced an immune response and antibodies with specific specificity were screened using conventional point-break analysis. The antibody was analyzed by Western analysis to identify the insulin-like growth factor I in Taiwan's Hongyu chicken blood 16 1308929. After separating the protein in the serum by the above-mentioned SDS polyacrylamide colloidal gel electrophoresis, the protein on the colloid was transferred. To nitrocellulose or polyvinylidene fluoride (PVDF) membrane, in blocking solution (100 ml washing buffer (2 〇 mmol of tris(hydroxymethyl) aminomethane hydrochloride pH 7.4, The membrane was blocked for one hour in 500 millimoles of sodium chloride, 0.05% Tween-20, and 3% gelatin and then washed with wash buffer. The above multi-drug antibody diluted with washing buffer and 1% gelatin was added to bind to the membrane, followed by the addition of alkaline phospholyase to the secondary of anti-rabbit immunoglobulin G (alkaline ph〇sphatase e() njugated anti-rabbiUgG) The antibody binds to the multi-strain antibody, and the binding antigen-determining region includes at least the sequence 丨 (4) acid moved to the alkaline phosphatase developing solution to perform snoring and scanning the laser densitometer to quantify the film after the egg* air-drying development And the body of the high-yielding egg group of Taiwan red plum soil Wei 1°, please refer to Figure 1 'The anti-object is about i3.7kDa and one percent of the serum is identified as having - precursor growth factor ι (the third phase of the electro-transformer, t from f A^7.GkDa
弟1道、第2道與第5道）。 實例三篩選高產蛋土雞 在兩個不同農場（育種t+a 紅羽土雞、來亨蛋雞以及△灣：1與育種農場2)飼養商用 在產蛋雞隻生長期的二:羽土雞的產蛋母雞。 26、27、32週收集全血以製備^18:19、2G、22、23、 測根據以下步驟測試禽·、。藉由酵素免疫吸附檢 量。 員”的類胰島素生長因子I含 1308929 在以合適抗原包覆孔盤的步驟中，取150微升/孔之 已稀釋的蛋白質標準/血清加入96孔盤中，置於4°C隔夜 培養。蛋白質標準是以包覆缓衝液（4.3克碳酸氫鈉、5.3 克碳酸納並以蒸餾水pH9.4加滿至1公升）連續稀釋並且 適當地稀釋血清，倒掉96孔盤中的液體去除未結合的抗原 並以200微升的清洗緩衝液（8.0克氯化鈉、1.42克磷酸氫 二鈉（Na2HP04.2H20)、0.2克磷酸氫二鉀、0.2克氯化 鉀、1毫升吐溫20並以蒸餾水pH 7.4加滿至1公升）清洗 • 孔盤，接著利用200微升的阻斷液（8.0克氯化鈉、1.42克 磷酸氫二鈉、0.2克磷酸氳二鉀、0.2克氣化鉀、10克胎 牛血清蛋白（bovine serum albumin (BSA) fraction V)並以蒸餾水 pH7.4加滿至1公升）在室溫反應45分鐘以阻斷非專一性 結合，並如上述清洗孔盤。 加入以抗體稀釋液（8.0克氯化鈉、ι·42克磷酸氫二 鈉、0.2克磷酸氫二鉀、0.2克氯化鉀、1〇克胎牛血清蛋 白並以蒸餾水pH 7.4加滿至1公升）稀釋的1〇〇微升兔子 參 多株抗體並在37。(：培養箱内培養45分鐘，重複上述清洗步 驟。製備接有鹼性磷解酶或辣根過氧化酶的抗兔子免疫球 蛋白G二級抗體並以抗體稀釋液稀釋，再加入1〇〇微升稀 釋後的抗兔子免疫球蛋白G二級抗體並在37°C培養箱内培 養30分鐘’重複上述清洗步驟。接著’再加入丨⑻微升的 受質液（四曱基聯苯胺（tetramethyl benzidine))避光至於37〇c 培養箱内20分鐘，加入1〇〇微升終止液（16毫升ιΝ鹽酸 以蒸餾水加滿至1公升）終止反應，並在酵素免疫吸附檢 1308929 測盤讀取機讀取孔盤以判斷在波長㈣奈米的光學密度 =pt:l Density)。由所測量的蛋白質標準取得標準曲線， ^據標準曲線計算血清内的蛋白質濃度。表2列舉了红 =雞與料雞在列時點収集血㈣ 長 因子I平均含量。 网矛、王负 表2 R( 紅羽土 雞） 週 數 隻 平均 3 標 準 數 偏 差 8 < Λ 14Brother 1st, 2nd and 5th). Example 3 Screening of high-yield egg-laying chickens in two different farms (breeding t+a red-feathered chicken, Laiheng laying hen and △wan: 1 and breeding farm 2) breeding commercials in the growing season of laying hens: Chicken laying hens. Whole blood was collected at 26, 27, and 32 weeks to prepare ^18:19, 2G, 22, and 23, and the test was carried out according to the following steps. Immunosorbent assay by enzyme. Insulin-like growth factor I contains 1308929. In the step of coating the well plate with a suitable antigen, 150 μl/well of the diluted protein standard/serum was added to a 96-well plate and placed at 4 ° C overnight. The protein standard was serially diluted with a coating buffer (4.3 g sodium bicarbonate, 5.3 g sodium carbonate and up to 1 liter in distilled water pH 9.4) and the serum was diluted appropriately, and the liquid in the 96-well dish was removed to remove unbound. Antigen with 200 μl of wash buffer (8.0 g of sodium chloride, 1.42 g of disodium hydrogen phosphate (Na2HP04.2H20), 0.2 g of dipotassium hydrogen phosphate, 0.2 g of potassium chloride, 1 ml of Tween 20 and Distilled water pH 7.4 to 1 liter) Wash • Well plate, then use 200 μl of blocking solution (8.0 g of sodium chloride, 1.42 g of disodium hydrogen phosphate, 0.2 g of dipotassium phosphate, 0.2 g of potassium hydride, 10 g of fetal bovine serum albumin (BSA) fraction V and topped up to 1 liter with distilled water pH 7.4) was reacted at room temperature for 45 minutes to block non-specific binding and the wells were washed as described above. Antibody dilution (8.0 g sodium chloride, ι·42 g disodium hydrogen phosphate, 0.2 1 liter microliter of rabbit ginseng antibody diluted in di-potassium hydrogen phosphate, 0.2 gram potassium chloride, 1 gram of fetal bovine serum albumin and 1 liter of distilled water pH 7.4 and added at 37. (: incubator The above washing step was repeated for 45 minutes, and an anti-rabbit immunoglobulin G secondary antibody with alkaline phosphatase or horseradish peroxidase was prepared and diluted with the antibody dilution, and then diluted 1 〇〇 microliter. Anti-rabbit immunoglobulin G secondary antibody and incubated in a 37 ° C incubator for 30 minutes' repeat the above washing step. Then add '8 (8) microliters of the receptor solution (tetramethyl benzidine) Light up to 37 〇c incubator for 20 minutes, add 1 〇〇 microliter of stop solution (16 ml of ιΝHCl to 1 liter of distilled water) to stop the reaction, and read the well in the enzyme immunosorbent 1308929 disk reader Disk to determine the optical density at wavelength (four) nm = pt: l Density). The standard curve was obtained from the measured protein standard, and the protein concentration in the serum was calculated according to the standard curve. Table 2 lists the red = chicken and chicken in the Collecting blood at the time of the column (4) I average content. Net spear, Wang negative Table 2 R (red feather chicken) weeks only average 3 standard number deviation 8 < Λ 14
60 60 60 22.4 - · 肽免毫疫彳所她對於抗原胜 18 22 26 32 ^縣㈣雞射，血義胰島 Γίϊ週較高，表示雞隻達到產蛋的成熟期丄方 較高I雞^血義胰島素生長因子1濃度在19至23週 匕不來予雞比紅羽土雞較早達到成熟期。 、另外紀錄每隻母雞在55週内所彦的魏疋m + 劂的頬胰島素生長因子1含量進行統計分檢 19 1308929 列，對於不同雞隻，相關係數（correlation coefficient)測量兩 個變數（產蛋總數與血清類胰島素生長因子I濃度）之間 正相關的程度。 表3 血清IGF-I濃度a與55週總產蛋數之相關係數b 採血時間點（週齡） 血清IGF-I濃度a與55週總產蛋數之相關係數b 採血時間點（週齡） 隻數 ⑹ 55週平均 產蛋數 (M±SD) 8 14 18 19 20 管理模式一 商用 72 60.7±43.0 0.014 0.073 - - 來亨 蛋雞 20 175.3±50.7 -0.430 0.410 - 0.190 - 管理模式二 紅羽 土雞 60 83.7±26.7 0.158 0.244 0.254* - - 來亨 蛋難 19 153.5±42.9 0.077 0.070 - 0.297 - 隻數 55週平均 22 23 26 27 32 ⑻ 產蛋數 20 1308929 (M 士 SD) 管理模式一 商用 72 60.7 士 43.0 - 0.617 - 0.470 0.422 來亨 蛋雞 20 175.3±50.7 - 0.337 - 0.729 管理模式二 红羽 土雞 60 83.7±26.7 0.152 - 0.342 - 0.373 來亨 蛋雞 19 153.5±42.9 - 0.357 - - 0.632 a以酵素免疫吸附檢測所分析 b相關係數(r) #差異顯著程度ρ<〇.〇1 接近1的高相關係數表示企清類胰島素生長因子I濃 度與雞隻產蛋數非常有關，如表3所顯示，多數的土雞不 論其育種農場均顯示血清類胰島素生長因子I濃度與55週 • 所產雞蛋總數之間具有高相關係數，因此本發明提供一種 有效篩選高產蛋禽類，特別是篩選高產蛋土雞的方法。 實例四使用PONT生物感測器進行田間測試 在產蛋土雞生長期的25至37週（25-37wk)收集全血以 製備血清檢體。將5微升的血清檢體與995微升的稀釋液 加入1.5毫升的微量離心管混合備用，取1〇〇微升已稀釋 的檢體，透過收集窗加入含實例二所合成之兔子多株抗體 21 1308929 的試劑條’使檢料抗狀鼓_分 =器中的掃描_土雞血清中的類 接著，在一共1197個血清檢體中， 騰島素生長因子1含量濃度最低的⑽血清類 9°%血清檢體進行產蛋性能的分析。如圖二=二二 出類胰島素生長因子1含量濃度最低的1。% h檢體與其他90%Α清檢體之間的產蛋率差異。而由此 田間測試的分析結果可判_胰島素生長目子〗含量濃度 較低的土雞其產蛋率籠較同群内其他场較低，因此本 發明提供了一種篩選高產蛋禽類的有效方法。 實例五使用酵素免疫吸附檢測進行田間測試 在產蛋土雞生長期的25至50週(25_5〇wk)收集全血以製 備血清檢體。根據實例三所述之酵素免疫吸附檢測步驟檢 測土雞血清檢體中的類胰島素生長因子j含量。 接者，在一共1560個血清檢體中，由土雞群中選取類 胰島素生長因子I含量濃度最低的1〇〇/〇血清檢體與其他 90%血清檢體進行產蛋性能的分析。此外，此實例中的25 至27週的產蛋率係根據實例四試驗結果以下列公式所推算 而來： 濃度最低的 10% 檢體：Υ=-〇.〇1〇3Χ2 + 〇.666Χ-10.124 其他濃度的 90% 檢體：Y = -0.052X2 + 0.3284X - 4.483 其中，Y為產蛋率，X為週齡。例如，在此實例中類 22 1308929 胰島素生長因子I含量濃度最低的10%檢體，26週的產蛋 率為： Y- -0.0103 (26)2 + 0.666 (26) - 10.124 = 0.22 並以此類推類在胰島素生長因子I含量濃度最低的 10%檢體與其餘90%檢體在25至27週的產蛋率。 如圖三所示，於29週前同樣可觀察出類胰島素生長因 子I含量濃度最低的10%檢體與其他90%檢體之間的產蛋 率差異。而由此田間測試的分析結果更進一步證明類胰島 φ 素生長因子I含量濃度較低的土雞其產蛋率明顯較同群内 其他土雞較低，因此本發明提供了一種篩選高產蛋禽類的 有效方法。 熟諳本項技藝之人士應即暸解，可對前述各具體實施 ' 例進行修改而不致悖離其廣義發明概念。從而應瞭解本發 ' 明並不限於本揭之各項特定具體實施例，而係為涵蓋歸屬 如後載申請專利範圍中所定義之本發明精神及範疇的修改 項目。 23 1308929 【圖式簡單說明】 圖一所示為聚丙烯醯胺凝膠電泳的膠體圖像顯示類胰 島素生長因子I的蛋白條帶。圖中標示1、2、5處為高 產蛋雞血清，標示3、4、6處為低產蛋雞血清。 圖二所示為一曲線圖顯示使用PONT生物感測器進行 田間測試的分析結果。 • 圖三所示為一曲線圖顯示使用酵素免疫吸附檢測進行 田間測試的分析結果。 2460 60 60 22.4 - · The peptide is free of plague. She is the winner of the antigen. 18 22 26 32 ^ County (four) chicken shot, bloody islet Γ ϊ ϊ high, indicating that the chicken reaches the maturity of laying eggs, higher I chicken ^ The blood insulin-like growth factor 1 concentration did not come to the chicken at 19 to 23 weeks earlier than the red-feathered chicken to reach maturity. In addition, the 頬Insulin Growth Factor 1 content of Wei 疋 m + 每 of each hen in each of the hens was recorded for statistical analysis of 19 1308929. For different chickens, the correlation coefficient was measured for two variables ( The degree of positive correlation between the total number of eggs produced and the serum insulin-like growth factor I concentration). Table 3 Correlation coefficient between serum IGF-I concentration a and total egg production at 55 weeks b Blood collection time point (week age) Correlation coefficient between serum IGF-I concentration a and total number of eggs produced in 55 weeks b Blood collection time point (week age) Only (6) 55 weeks average number of eggs laid (M ± SD) 8 14 18 19 20 Management mode a commercial 72 60.7 ± 43.0 0.014 0.073 - - Lai Hen hens 20 175.3 ± 50.7 -0.430 0.410 - 0.190 - Management mode two red feather soil Chicken 60 83.7±26.7 0.158 0.244 0.254* - - Lai Hen Egg Difficult 19 153.5±42.9 0.077 0.070 - 0.297 - Only 55 Weekly Average 22 23 26 27 32 (8) Number of Eggs 20 1308929 (M SSD) Management Mode One Commercial 72 60.7 士士43.0 - 0.617 - 0.470 0.422 来亨蛋鸡20 175.3±50.7 - 0.337 - 0.729 Management mode two red feather chicken 60 83.7±26.7 0.152 - 0.342 - 0.373 Lai Hen hen 19 153.5±42.9 - 0.357 - - 0.632 a Analysis of b correlation coefficient (r) by enzyme immunosorbent assay # difference significant degree ρ<〇.〇1 The high correlation coefficient close to 1 indicates that the concentration of insulin-like growth factor I in Qiqing is very related to the number of eggs laid by chickens, as shown in Table 3. It shows that most of the chickens are no matter Farm showed serum concentrations of insulin-like growth factor I and 55 weeks • produced having a high correlation between the total number of eggs, the present invention thus provides a highly efficient screening of egg poultry, in particular chicken egg of high screening method. Example 4 Field testing using a PONT biosensor Whole blood was collected at 25 to 37 weeks (25-37 wk) in the laying period of the laying chicken to prepare a serum sample. Mix 5 μl of the serum sample with 995 μl of the diluted solution into a 1.5 ml microcentrifuge tube, and take 1 μl of the diluted sample. Add the rabbits synthesized in Example 2 through the collection window. The reagent strip of antibody 21 1308929 'make the test against the drum _ points = the scan in the device _ the class in the chicken serum, then, in a total of 1197 serum samples, the lowest concentration of the growth factor 1 (10) serum Analysis of egg production performance by a 9°% serum sample. As shown in Figure 2 = 2, the concentration of insulin-like growth factor 1 is the lowest. Difference in egg production between the %h specimen and the other 90% sputum specimens. Therefore, the analysis result of the field test can judge that the egg production rate of the chicken with lower concentration of insulin growth is lower than that of other fields in the same group, so the present invention provides an effective screening for high-yielding eggs and poultry. method. Example 5 Field test using enzyme immunosorbent assay Whole blood was collected for 25 to 50 weeks (25_5 〇 wk) in the laying period of the laying chicken to prepare a serum sample. The content of insulin-like growth factor j in the serum samples of the chickens was examined according to the enzyme immunosorbent detection step described in Example 3. In a total of 1560 serum samples, the 1〇〇/〇 serum samples with the lowest concentration of insulin-like growth factor I were selected from the chicken flocks and the other 90% serum samples were analyzed for egg production performance. In addition, the 25- to 27-week egg production rate in this example is based on the results of the four test results using the following formula: 10% of the lowest concentration. Sample: Υ=-〇.〇1〇3Χ2 + 〇.666Χ- 10.124 90% of other concentrations. Sample: Y = -0.052X2 + 0.3284X - 4.483 where Y is the egg production rate and X is the age of the week. For example, in this example, class 22 1308929 has the lowest 10% concentration of insulin growth factor I, and the 26-week egg production rate is Y- -0.0103 (26) 2 + 0.666 (26) - 10.124 = 0.22 and The analogy was found in the lowest concentration of insulin growth factor I in 10% of the samples and the remaining 90% of the samples at 25 to 27 weeks of egg production. As shown in Figure 3, the difference in egg production between the 10% sample with the lowest concentration of insulin-like growth factor I and the other 90% was observed before 29 weeks. The analysis results of the field test further confirmed that the laying rate of the chickens with lower concentration of the islet-like growth factor I was significantly lower than that of other chickens in the same group, so the present invention provides a screening for high-yielding eggs. An effective method for poultry. Those skilled in the art should understand that the foregoing specific embodiments may be modified without departing from the broad inventive concept. It is to be understood that the present invention is not limited to the specific embodiments of the present invention, but is intended to cover modifications of the spirit and scope of the invention as defined in the appended claims. 23 1308929 [Simple description of the diagram] Figure 1 shows a colloidal image of insulin-like gelatin electrophoresis showing a protein band of insulin-like growth factor I. In the figure, 1, 2, and 5 are high-production laying hen serum, and 3, 4, and 6 are low-yielding laying hen serum. Figure 2 shows a graph showing the results of an analysis using a PONT biosensor for field testing. • Figure 3 shows a graph showing the results of a field test using enzyme immunosorbent assay. twenty four