基于單域重鏈抗體的天然噬菌體文庫(kù)的構(gòu)建及抗DON適配體的淘選
發(fā)布時(shí)間：2019-06-17 19:18
【摘要】：重鏈抗體是一種存在于駱駝、鯊魚等動(dòng)物中天然缺失輕鏈的抗體,僅由其可變區(qū)組成的抗體稱為單域重鏈抗體。單域重鏈抗體分子量小(約15 kDa),具有易表達(dá)、高水溶性、耐高溫、耐極度pH值等獨(dú)特性質(zhì),目前已廣泛用于基礎(chǔ)研究、醫(yī)學(xué)診斷和檢測(cè)、抗體藥物開發(fā)等領(lǐng)域。在食品安全檢測(cè)領(lǐng)域單域重鏈抗體也展現(xiàn)出廣闊的發(fā)展前景。 脫氧雪腐鐮刀菌烯醇(deoxynivalenol,DON),又名嘔吐毒素(vomitoxin,VT),屬于單端孢霉烯族真菌毒素,主要是由某些鐮刀菌產(chǎn)生。該毒素常出現(xiàn)于谷物及其加工產(chǎn)品中,許多國(guó)家和地區(qū)都檢測(cè)到了DON的污染。DON會(huì)引起動(dòng)物增重下降、食欲減退、營(yíng)養(yǎng)吸收率降低,并影響免疫功能。目前大部分國(guó)家對(duì)食品、谷物、飼料中的DON含量都做了嚴(yán)格規(guī)定。 免疫學(xué)檢測(cè)方法具有靈敏、快速等特點(diǎn),適合大批量樣本的檢測(cè)。現(xiàn)有的DON免疫學(xué)檢測(cè)方法大多采用傳統(tǒng)的抗體制備技術(shù)(多克隆抗體和單克隆抗體),需要多次反復(fù)免疫動(dòng)物,制備過程繁瑣,來(lái)源有限,生產(chǎn)成本較高。以抗體庫(kù)技術(shù)為代表的第三代基因工程抗體技術(shù)為快速、低成本的獲取和生產(chǎn)抗體提供了強(qiáng)有力的支撐。 本研究采用未經(jīng)免疫的健康羊駝(Lama pacos)的免疫細(xì)胞作為實(shí)驗(yàn)材料,根據(jù)重鏈抗體保守序列設(shè)計(jì)引物,建立基于重鏈抗體可變區(qū)基因的天然噬菌體展示文庫(kù),并以DON-MBSA人工抗原為配基,對(duì)構(gòu)建的文庫(kù)進(jìn)行了淘選,主要研究結(jié)果如下： 1以羊駝(Lama pacos)外周血為起始材料,分別采用半巢式PCR法和巢式PCR法構(gòu)建了兩個(gè)單域重鏈抗體初級(jí)文庫(kù)SNAL和NAL,兩者的實(shí)際庫(kù)容量均達(dá)到107,經(jīng)輔助噬菌體救援后獲得噬菌體展示文庫(kù)SNA-PDL和NA-PDL,滴度達(dá)1013cfu/mL。文庫(kù)多樣性分析結(jié)果顯示,文庫(kù)具有良好的多樣性,可以用于后續(xù)淘選。 2通過三輪淘選,從噬菌體文庫(kù)SNA-PDL中淘選出了三類能與DON特異性結(jié)合的克隆,分別為DON-binder 1:B9、B10、B11、B13、C3、C8、C10、C15; DON-binder 2:C11; DON-binder 3:A4、A9。間接phage-ELISA檢測(cè)結(jié)果顯示,前兩類克隆的OD450測(cè)定值可達(dá)后一類克隆OD450值的7～10倍,提示親和力較高。從天然噬菌體文庫(kù)NA-PDL中淘選獲得了兩種可以與DON-MBSA結(jié)合的單域重鏈抗體,其中一種可以與DON特異性結(jié)合。 3在大腸桿菌表達(dá)系統(tǒng)中對(duì)重組噬菌粒pHEN-B9進(jìn)行誘導(dǎo)表達(dá),SDS-PAGE電泳結(jié)果顯示,在誘導(dǎo)培養(yǎng)上清中存在預(yù)期大小的蛋白(約50 kDa)。競(jìng)爭(zhēng)ELISA法分析誘導(dǎo)表達(dá)上清,結(jié)果表明誘導(dǎo)表達(dá)上清可以抑制抗DON單克隆抗體與人工抗原的結(jié)合,提示培養(yǎng)上清中的表達(dá)產(chǎn)物能與DON發(fā)生特異性結(jié)合,有望替代抗DON單克隆抗體。 本研究的主要?jiǎng)?chuàng)新之處： 1采用針對(duì)羊駝(Lama pacos)重鏈抗體設(shè)計(jì)的引物,分別以巢式PCR法和半巢式PCR法擴(kuò)增羊駝重鏈抗體可變區(qū)全套基因,構(gòu)建了兩個(gè)天然噬菌體文庫(kù)SNA-PDL和NA-PDL,該文庫(kù)可以作為通用的技術(shù)平臺(tái),重復(fù)用于多種抗原的篩選。 2從天然噬菌體文庫(kù)SNA-PDL中淘選獲得了三種可以與DON特異結(jié)合的克隆,選取其中一種結(jié)合力較強(qiáng)的克隆在大腸桿菌中表達(dá),結(jié)果表明,能夠表達(dá)出具有DON結(jié)合活性的蛋白,有望將其應(yīng)用于DON免疫學(xué)檢測(cè)方法的建立。從天然噬菌體文庫(kù)NA-PDL中淘選獲得了一種可以與DON特異性結(jié)合的單域重鏈抗體。
[Abstract]:The heavy chain antibody is an antibody that is naturally missing a light chain in an animal such as a camel, a shark, and the like, and an antibody composed of only a variable region thereof is referred to as a single-domain heavy chain antibody. The single-domain heavy chain antibody has a small molecular weight (about 15 kDa), and has unique properties such as easy expression, high water solubility, high temperature resistance, extreme pH value, and the like, and is widely used in the fields of basic research, medical diagnosis and detection, antibody drug development and the like. The single-domain heavy chain antibody in the field of food safety detection also has a wide development prospect. Deoxynivalenol (DON), also known as a Vomitoxin (VT), belongs to a single-end trichoderma mycotoxin, which is mainly caused by certain fusarium The toxin is often present in the grain and its processed products, and DON is detected in many countries and regions Contamination. DON causes decreased weight gain, decreased appetite, reduced nutritional absorption, and immune response In most countries, the DON content in food, grain and feed is strictly controlled. The immunological detection method is sensitive, rapid and the like, and is suitable for mass production. The conventional DON immunological detection method mainly adopts the traditional antibody preparation technology (polyclonal antibody and monoclonal antibody), The third-generation genetic engineering antibody technology, which is represented by the antibody library technology, is a rapid, low-cost acquisition and production antibody. In this study, the immune cells of the unimmunized healthy alpaca (Lama pacos) were used as the experimental material. The primers were designed according to the conservative sequence of the heavy chain antibody, and a natural phage display library based on the variable region gene of the heavy chain was established and the DON-MBS was used. A artificial antigen is a ligand, and the constructed library is subjected to panning, The main results are as follows:1. The peripheral blood of the Lama pacos is used as the starting material, and the two single-domain heavy chain antibody primary libraries, NAL and NAL, are constructed by using a semi-nested PCR method and a nested PCR method, respectively. The actual library capacity reached 107, and the SNA-PDL and NA-PDL, titer of the phage display library were obtained after the auxiliary phage rescue. The results of the analysis of the library diversity showed that the library had a good multi-function. Samples can be used for subsequent panning. Three types of clones capable of specifically binding to DON are selected from the phage library, SNA-PDL, by three-wheel panning, respectively: DON-finder 1: B9, B10, B11, B13, C3, C8, C10, C15; DON-Binder 2: C11; DON -Binder 3: A4, A9. The indirect phage-ELISA test results showed that the OD450 values of the first two types of clones can reach the last class of clone OD450 values Two single-domain heavy chain antibodies which can be combined with DON-MBSA are obtained from the natural phage library NA-PDL, and the single-domain heavy chain antibody can be combined with DON-MBSA, in that expression system of E. coli, the recombinant phagemid pHen-B9 is induce and expressed, and the SDS-PAGE electrophoresis result shows that in the culture supernatant, The protein (about 50 kDa) of the expected size was present. The expression of the supernatant was analyzed by competitive ELISA. The results showed that the expression of the supernatant could inhibit the binding of the anti-DON monoclonal antibody to the artificial antigen, suggesting that the expression product in the culture supernatant can be specific to DON. Sex-binding, promising alternative to anti-DO The main innovations of this study were:1. Using the primers designed for the heavy chain antibody of the alpaca (Lama pacos), the whole gene of the variable region of the heavy chain of the alpaca was amplified by a nested PCR method and a semi-nested PCR method. Two natural phage libraries, SNA-PDL and NA-PDL, the library Can be used as a general technical platform and is repeatedly used for screening a plurality of antigens. Three clones which can be specifically combined with DON are obtained from the SNA-PDL of the natural phage library, and one of the clones with strong binding force is selected to be in Escherichia coli. The results show that the combination of DON and DON can be expressed. The active protein is expected to be applied to the establishment of the DON immunological detection method. From the natural phage library, NA-PDL
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392
[Abstract]:The heavy chain antibody is an antibody that is naturally missing a light chain in an animal such as a camel, a shark, and the like, and an antibody composed of only a variable region thereof is referred to as a single-domain heavy chain antibody. The single-domain heavy chain antibody has a small molecular weight (about 15 kDa), and has unique properties such as easy expression, high water solubility, high temperature resistance, extreme pH value, and the like, and is widely used in the fields of basic research, medical diagnosis and detection, antibody drug development and the like. The single-domain heavy chain antibody in the field of food safety detection also has a wide development prospect. Deoxynivalenol (DON), also known as a Vomitoxin (VT), belongs to a single-end trichoderma mycotoxin, which is mainly caused by certain fusarium The toxin is often present in the grain and its processed products, and DON is detected in many countries and regions Contamination. DON causes decreased weight gain, decreased appetite, reduced nutritional absorption, and immune response In most countries, the DON content in food, grain and feed is strictly controlled. The immunological detection method is sensitive, rapid and the like, and is suitable for mass production. The conventional DON immunological detection method mainly adopts the traditional antibody preparation technology (polyclonal antibody and monoclonal antibody), The third-generation genetic engineering antibody technology, which is represented by the antibody library technology, is a rapid, low-cost acquisition and production antibody. In this study, the immune cells of the unimmunized healthy alpaca (Lama pacos) were used as the experimental material. The primers were designed according to the conservative sequence of the heavy chain antibody, and a natural phage display library based on the variable region gene of the heavy chain was established and the DON-MBS was used. A artificial antigen is a ligand, and the constructed library is subjected to panning, The main results are as follows:1. The peripheral blood of the Lama pacos is used as the starting material, and the two single-domain heavy chain antibody primary libraries, NAL and NAL, are constructed by using a semi-nested PCR method and a nested PCR method, respectively. The actual library capacity reached 107, and the SNA-PDL and NA-PDL, titer of the phage display library were obtained after the auxiliary phage rescue. The results of the analysis of the library diversity showed that the library had a good multi-function. Samples can be used for subsequent panning. Three types of clones capable of specifically binding to DON are selected from the phage library, SNA-PDL, by three-wheel panning, respectively: DON-finder 1: B9, B10, B11, B13, C3, C8, C10, C15; DON-Binder 2: C11; DON -Binder 3: A4, A9. The indirect phage-ELISA test results showed that the OD450 values of the first two types of clones can reach the last class of clone OD450 values Two single-domain heavy chain antibodies which can be combined with DON-MBSA are obtained from the natural phage library NA-PDL, and the single-domain heavy chain antibody can be combined with DON-MBSA, in that expression system of E. coli, the recombinant phagemid pHen-B9 is induce and expressed, and the SDS-PAGE electrophoresis result shows that in the culture supernatant, The protein (about 50 kDa) of the expected size was present. The expression of the supernatant was analyzed by competitive ELISA. The results showed that the expression of the supernatant could inhibit the binding of the anti-DON monoclonal antibody to the artificial antigen, suggesting that the expression product in the culture supernatant can be specific to DON. Sex-binding, promising alternative to anti-DO The main innovations of this study were:1. Using the primers designed for the heavy chain antibody of the alpaca (Lama pacos), the whole gene of the variable region of the heavy chain of the alpaca was amplified by a nested PCR method and a semi-nested PCR method. Two natural phage libraries, SNA-PDL and NA-PDL, the library Can be used as a general technical platform and is repeatedly used for screening a plurality of antigens. Three clones which can be specifically combined with DON are obtained from the SNA-PDL of the natural phage library, and one of the clones with strong binding force is selected to be in Escherichia coli. The results show that the combination of DON and DON can be expressed. The active protein is expected to be applied to the establishment of the DON immunological detection method. From the natural phage library, NA-PDL
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392
【參考文獻(xiàn)】
相關(guān)期刊論文 前3條
1 謝磊,孫建波,張世清,黃俊生;大腸桿菌表達(dá)系統(tǒng)及其研究進(jìn)展[J];華南熱帶農(nóng)業(yè)大學(xué)學(xué)報(bào);2004年02期
2 馮強(qiáng),楊s，
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