【摘要】：目的：?jiǎn)卧隼钏固鼐?Listeria monocytogenes，LM)是李斯特菌屬最重要的人和動(dòng)物致病菌，由于其具有低溫生長(zhǎng)的特性，因此是冷藏食品和即食食品最重要的致病菌之一。本文通過(guò)原核表達(dá)單增李斯特菌特異性膜表面蛋白的基因iap，actA，plcB，并分析其產(chǎn)物作為制備LM特異性膜表面蛋白單克隆抗體的可行性，為今后制備這些蛋白的單克隆抗體并建立單增李斯特菌的免疫學(xué)磁珠分離快速檢測(cè)方法的建立奠定基礎(chǔ)。方法：分別設(shè)計(jì)actA，plcB，iap基因引物，并以提取的單增李斯特菌DNA為模板進(jìn)行PCR擴(kuò)增，產(chǎn)物回收后，連接入pMD18-T克隆載體，轉(zhuǎn)化E.coliDH5α，提取質(zhì)粒并進(jìn)行雙酶切鑒定及測(cè)序鑒定。鑒定正確的的重組質(zhì)粒與表達(dá)載體PET28a(+)/PET32a（+）構(gòu)建重組表達(dá)質(zhì)粒，產(chǎn)物轉(zhuǎn)化E.coli BL21，經(jīng)含卡那霉素/氨芐青霉素的LB培養(yǎng)基篩選，挑取陽(yáng)性菌落并進(jìn)行測(cè)序。將鑒定正確的菌液ITPG誘導(dǎo)表達(dá)，超聲破碎菌體，SDS-PAGE對(duì)表達(dá)產(chǎn)物進(jìn)行可溶性表達(dá)分析，利用親和層析分離純化目的蛋白，運(yùn)用westernblotting和ELISA分析重組蛋白的免疫原性，將純化的p60蛋白免疫小鼠，制備鼠多克隆抗體，ELISA檢測(cè)效價(jià)以及交叉反應(yīng)性。結(jié)果：雙酶切及測(cè)序鑒定顯示，重組克隆載體及表達(dá)載體均構(gòu)建成功。分析誘導(dǎo)表達(dá)產(chǎn)物，ActA蛋白和p60蛋白為可溶性表達(dá)，plcB為包涵體表達(dá)。鎳離子親和層析純化，得到了純度較高的PlcB蛋白和p60蛋白，但是ActA沒(méi)有純化成功。免疫原性分析表明：p60蛋白和ActA蛋白與兔和鼠免疫血清均具有反應(yīng)性，plcB與兔免疫血清反應(yīng)良好。以重組表達(dá)的p60蛋白作為免疫原制備鼠多克隆抗體，3次免疫后效價(jià)達(dá)到1:8000，并且與屬內(nèi)威爾斯、英諾克菌存在微弱交叉反應(yīng)。結(jié)論：原核表達(dá)單增李斯特菌毒力因子基因actA，plcB，iap，，分析了所表達(dá)蛋白的免疫原性，其中重組p60蛋白對(duì)于家兔和小鼠均有較好的免疫原性，免疫小鼠后所得多抗與LM菌體特異性結(jié)合，這表明以其為靶點(diǎn)，制備LM特異性的膜表面單抗是可行的。
[Abstract]:Objective: Listeria monocytogenes (Listeria monocytogenes,LM) is the most important human and animal pathogen of listeria. Because of its low temperature growth characteristics, Listeria monocytogenes is one of the most important pathogenic bacteria in refrigerated food and ready-to-eat food. In this paper, the gene iap,actA,plcB, of Listeria monocytogenes specific membrane surface protein was expressed in prokaryotic cells and the feasibility of using its product as monoclonal antibody to LM specific membrane surface protein was analyzed. It lays a foundation for the preparation of monoclonal antibodies against these proteins and the establishment of a rapid method for the separation and detection of Listeria monocytogenes by immunological magnetic beads. Methods: actA,plcB,iap gene primers were designed, and the extracted Listeria monocytogenes DNA was used as template for PCR amplification. after the product was recovered, the product was ligated into pMD18-T clone vector and transformed into E.coliDH5 偽. The plasmid was extracted and identified by double enzyme digestion and sequencing. The correct recombinant plasmid and expression vector PET28a () / PET32a () were identified to construct the recombinant expression plasmid. The product was transformed into E.coli BL21, and screened by LB medium containing kanamycin / ampicillin. The positive colonies were selected and sequenced. The correct ITPG induced expression was identified, the expressed product was analyzed by SDS-PAGE, the target protein was isolated and purified by affinity chromatography, and the immunogenicity of the recombinant protein was analyzed by westernblotting and ELISA. Mouse polyclonal antibody was prepared by immunizing mice with purified p60 protein. The titer and cross-reaction of mouse polyclonal antibody were detected by ELISA. Results: double enzyme digestion and sequencing showed that the recombinant clone vector and expression vector were constructed successfully. The induced expression products were analyzed. ActA protein and p60 protein were soluble and plcB was expressed as inclusion body. High purity PlcB protein and p60 protein were obtained by nickel ion affinity chromatography, but ActA was not purified successfully. Immunogenicity analysis showed that p60 protein and ActA protein were reactive to rabbit and mouse immune serum, and plcB reacted well with rabbit immune serum. Mouse polyclonal antibody was prepared by using recombinant p60 protein as immunogen. The titer of mouse polyclonal antibody reached 1 鈮

本文編號(hào)：2484024