【摘要】：隨著科學(xué)技術(shù)的飛速發(fā)展，人們的生活水平也有了很大提高。特別是二戰(zhàn)后，人們的飲食結(jié)構(gòu)有了很大變化，肉類和油脂的消費(fèi)劇增是食物結(jié)構(gòu)中最大的變化。而與之相隨的是，糖尿病、心腦血管疾病、腫瘤等的發(fā)病率也飛速增多。中國已經(jīng)是肥胖人口增長最快的國家之一，是糖尿病患者人數(shù)居世界第二，Ⅱ型糖尿病發(fā)病率世界第一。研究表明，血漿中脂聯(lián)素水平與這些疾病的發(fā)生有密切關(guān)系，且脂聯(lián)素球狀區(qū)是其行使功能的主要結(jié)構(gòu)。故通過對脂聯(lián)素水平的測定可以為臨床診斷這些疾病及臨床用藥提供指導(dǎo)。本研究利用基因工程技術(shù)獲得具有生物活性的人脂聯(lián)素球狀區(qū)融合蛋白，利用其制備單克隆抗體，為建立人脂聯(lián)素免疫學(xué)檢測方法奠定基礎(chǔ)。 根據(jù)NCBI上公布的人脂聯(lián)素基因序列NM_004797.2，分析其密碼子并對其密碼子進(jìn)行優(yōu)化，然后人工合成人脂聯(lián)素基因球狀區(qū)序列，通過酶切，構(gòu)建表達(dá)載體pET41a-gAd。在大腸桿菌中表達(dá)，通過調(diào)節(jié)誘導(dǎo)劑濃度、誘導(dǎo)溫度和誘導(dǎo)時(shí)間進(jìn)行優(yōu)化，確立最佳誘導(dǎo)條件為：37℃，IPTG濃度0.1mmol/L，誘導(dǎo)表達(dá)6h。表達(dá)出人脂聯(lián)素球狀區(qū)融合蛋白，用Ni親和層析柱純化，采用包涵體復(fù)性技術(shù)對表達(dá)蛋白進(jìn)行復(fù)性，獲得具有生物活性的重組蛋白。把純化的人脂聯(lián)素球狀區(qū)蛋白以適當(dāng)劑量免疫BABL/C小鼠，取免疫效價(jià)較高小鼠的脾細(xì)胞和骨髓瘤細(xì)胞進(jìn)行細(xì)胞融合。經(jīng)HAT培養(yǎng)篩選、有限稀釋法克隆化，獲得能分泌抗人脂聯(lián)素球狀區(qū)單克隆抗體的細(xì)胞株。對細(xì)胞株進(jìn)行擴(kuò)大培養(yǎng)，收集雜交瘤細(xì)胞，，接種BABL/C小鼠腹腔誘生腹水，并對腹水進(jìn)行純化，經(jīng)SDS-PAGE分析其純度，并鑒定其亞型。 本研究成功構(gòu)建了pET41a-gAd載體，能高效表達(dá)出具有生物活性的人脂聯(lián)素球狀區(qū)融合蛋白；該蛋白分子量約為45kD，經(jīng)純化后純度為90%。用此蛋白成功制備備單克隆抗體。獲得了人脂聯(lián)素球狀區(qū)單克隆抗體細(xì)胞株1株，腹水純化后單抗純度為91%，所獲的單克隆抗體亞型為IgG1。
[Abstract]:With the rapid development of science and technology, people's living standards have been greatly improved. Especially after World War II, people's dietary structure has changed greatly. Consumption of meat and fat is the biggest change in food structure. With it, the incidence of diabetes, cardiovascular and cerebrovascular diseases, tumors and so on also increased rapidly. China has one of the fastest-growing obese people, the world's second largest number of people with diabetes, and the world's highest incidence of type 2 diabetes. The results showed that the level of adiponectin in plasma was closely related to the occurrence of these diseases, and adiponectin globular region was the main structure of its function. Therefore, the determination of adiponectin level can provide guidance for clinical diagnosis of these diseases and clinical use of drugs. In this study, human adiponectin globular fusion protein with biological activity was obtained by genetic engineering technique, and monoclonal antibody was prepared, which laid a foundation for the establishment of immunologic detection method of human adiponectin. The codon of human adiponectin gene was analyzed and optimized according to the published human adiponectin gene sequence NM_004797.2, in NCBI. Then the spherical region sequence of human adiponectin gene was synthesized and the expression vector pET41a-gAd. was constructed by restriction enzyme digestion. The expression in E. coli was optimized by adjusting the concentration of inducer, inducing temperature and inducing time. The optimal induction conditions were 37 鈩

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