本文選題：錦鯉皰疹病毒 + ORF68基因��； 參考：《吉林農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：KHVD給世界多個(gè)國(guó)家的鯉魚及錦鯉養(yǎng)殖業(yè)造成了嚴(yán)重的經(jīng)濟(jì)損失,引起這種現(xiàn)象主要是由于沒有快速安全有效的診斷方法,不能盡早發(fā)現(xiàn)及時(shí)診斷,應(yīng)用單克隆抗體技術(shù),可以在發(fā)病初期進(jìn)行診斷盡早發(fā)現(xiàn)問題及時(shí)解決問題。單克隆抗體技術(shù)應(yīng)用于錦鯉皰疹病毒病起步較晚,目前國(guó)外只有3篇相關(guān)報(bào)道,國(guó)內(nèi)仍然沒有成功案例,因此本研究致力于篩選出抗錦鯉皰疹病毒的單克隆抗體,推動(dòng)鯉魚的健康生長(zhǎng),對(duì)于我國(guó)鯉魚養(yǎng)殖具有重大的意義。以本實(shí)驗(yàn)室分離保存的KHV-CJ株DNA為模板,采用PCR擴(kuò)增技術(shù),分別擴(kuò)增出753 bp、897bp和1500 bp大小的ORF68基因片段,將三段基因插入到pMD18-T載體中,通過(guò)PCR和雙酶切鑒定后,確定克隆質(zhì)粒pMD18T-753、pMD18-T-807和pMD18-T-1500構(gòu)建成功。經(jīng)BLAST比對(duì),獲得的目的基因片段同源性可達(dá)到100%。分別用三段基因構(gòu)建重組表達(dá)質(zhì)粒,轉(zhuǎn)入BL21表達(dá)菌中,經(jīng)IPTG誘導(dǎo)后對(duì)重組蛋白進(jìn)行可溶性分析表明,本實(shí)驗(yàn)重組蛋白可以在大腸桿菌包涵體中和可溶性形式同時(shí)存在,分子量分別為45.61kDa、47.59kDa和73kDa。采用帶有His標(biāo)記的Ni離子層析柱進(jìn)行純化,經(jīng)測(cè)定重組蛋白濃度分別為:重組蛋白pET32a-753:0.46mg/ml,重組蛋白pET32a-807:0.57mg/ml,重組蛋白pET32a-1500:0.51mg/ml。按照免疫程序進(jìn)行小鼠免疫,ELISA檢測(cè)其免疫后的血清效價(jià),將免疫小鼠脾細(xì)胞與SP2/0細(xì)胞融合,經(jīng)HAT條件培養(yǎng)基篩選后,使用ELISA法檢測(cè)培養(yǎng)基上清效價(jià),初步篩選確定陽(yáng)性克隆后進(jìn)行多次篩選,根據(jù)OD450nm值,在其中篩選出表達(dá)量較高的細(xì)胞株,進(jìn)行有限稀釋篩選出的陽(yáng)性細(xì)胞擴(kuò)大培養(yǎng)后,注射小鼠腹腔,制備腹水。對(duì)制備的腹水進(jìn)行反應(yīng)原性、類別鑒定。結(jié)果表明:通過(guò)對(duì)單克隆細(xì)胞株培養(yǎng)基上清進(jìn)行初次檢測(cè)選擇出227株陽(yáng)性單克隆細(xì)胞株,進(jìn)行多次檢測(cè)后篩選出較穩(wěn)定15株陽(yáng)性結(jié)果,根據(jù)OD450nm值,在其中選出5株OD450nm值較高的,分別命名為:807-1D3、807-1D6、807-3E5、1500-3D2、1500-4G2,最終篩選出1株較穩(wěn)定的陽(yáng)性株:807-1D6,其抗體類別為IgG1,通過(guò)Western-blot確定其能識(shí)別KHV重組蛋白pET32a-807。
[Abstract]:KHVD has caused serious economic losses to carp and koi culture in many countries in the world. This phenomenon is mainly due to the lack of rapid, safe and effective diagnostic methods, the lack of prompt diagnosis as early as possible, and the application of monoclonal antibody technology. Can be diagnosed in the early stage of diagnosis as early as possible to solve the problem. The application of monoclonal antibody technology to koi herpesvirus disease started late. At present, there are only three related reports in foreign countries, but there are still no successful cases in China. Therefore, this study is devoted to screening monoclonal antibodies against koi herpesvirus. It is of great significance to promote the healthy growth of carp in China. Using the DNA of the KHV-CJ strain isolated and preserved in our laboratory as template, the ORF68 gene fragments of 753 BP, 897 BP and 1500 BP were amplified by PCR amplification technique. The three fragments were inserted into the pMD18-T vector and identified by PCR and double enzyme digestion. The cloned plasmid pMD18T-753pMD18-T-807 and pMD18-T-1500 were successfully constructed. The homology of the target gene fragment obtained by BLAST was 100%. The recombinant expression plasmids were constructed with three segments of genes and transferred into BL21 expression bacteria. The soluble analysis of the recombinant protein induced by IPTG showed that the recombinant protein could exist simultaneously in the inclusion body and soluble form of E. coli. The molecular weights were 45.61kDa 47.59kDa and 73kDa. respectively. The concentration of recombinant protein pET32a-753: 0.46 mg / ml, recombinant protein pET32a-807: 0.57 mg / ml, recombinant protein pET32a-1500: 0.51mg / ml were determined by using Ni ion chromatography column labeled with His. The results showed that the recombinant protein pET32a-753: 0.46 mg / ml, pET32a-807: 0.57 mg / ml, and pET32a-1500: 0.51 mg / ml, respectively. The serum titers of immunized mice were detected by Elisa according to the immune procedure. The spleen cells of immunized mice were fused with SP2/0 cells. The supernatant titers were detected by ELISA method after screening by HAT conditioned medium. According to the OD450nm value, the cell lines with high expression were screened. After the expansion of the positive cells selected by limited dilution, ascites were prepared by injecting mice intraperitoneally. The reactivity and classification of the prepared ascites were identified. The results showed that 227 positive monoclonal cell lines were selected from the supernatant of the culture medium for the first time, and 15 positive strains were screened after repeated tests. According to the OD450nm value, 5 of them were selected with higher OD450nm value. They were named as: 807-1D3807-1D6807-3E5A1500-3D21500-4G2. Finally, a stable positive strain: 807-1D6 was selected and its antibody class was IgG1. The recombinant KHV protein pET32a-807 was identified by Western-blot.
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S941.41

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