腦鈉肽單克隆抗體的制備及初步臨床應(yīng)用研究
發(fā)布時(shí)間：2019-02-16 21:32
【摘要】：目的: 心功能衰竭的早期診斷與干預(yù)在心臟疾患的診治中非常重要。腦鈉肽(BNP)作為一種主要來源于心室肌細(xì)胞的循環(huán)激素,在心室壁張力升高時(shí)迅速、大量分泌,其血漿濃度與心功能的相關(guān)性已被廣泛認(rèn)識(shí)。但目前國內(nèi)檢測BNP濃度靠進(jìn)口國外的各種不同試劑盒,每種試劑盒檢測范圍及參考值不同,結(jié)果亦不盡相同,缺乏可比性,且價(jià)格昂貴。為使BNP檢測國產(chǎn)化,本研究制備了BNP單克隆抗體,初步用于正常人及心臟病患者的血漿BNP濃度檢測,以評(píng)價(jià)自制BNP單抗檢測所得的BNP濃度與心功能的關(guān)系,為進(jìn)一步研制國產(chǎn)BNP單抗試劑盒奠定基礎(chǔ)。 方法: 1.根據(jù)GenBank中檢索到的人BNP成熟蛋白序列,拼接BNP的基因。以原核表達(dá)系統(tǒng)pET32構(gòu)建pET32a.BNP重組質(zhì)粒,經(jīng)誘導(dǎo)提取純化,制備人Trx-BNP融合蛋白。 2.以Trx-BNP融合蛋白作抗原,免疫小鼠,獲得分泌BNP抗體的雜交瘤細(xì)胞株。將雜交瘤細(xì)胞接種于小鼠腹腔,進(jìn)行BNP單克隆抗體的大量制備。檢測雜交瘤細(xì)胞的培養(yǎng)上清抗體效價(jià)及腹水抗體效價(jià)。以Western blot及免疫組化檢測制備的BNP抗體特異性。 3.用制備的BNP單克隆抗體以ELISA間接法檢測正常人及各級(jí)心功能的心臟病患者的血漿BNP濃度。正常人20例,心臟病患者92例,后者按紐約心臟病學(xué)會(huì)(NYHA)心功能分級(jí)分為:心功能Ⅰ級(jí)(24例)、Ⅱ級(jí)(23例)、Ⅲ級(jí)(20例)、Ⅳ級(jí)(25例),同時(shí)經(jīng)超聲心動(dòng)圖評(píng)價(jià)心臟收縮(EF值)及舒張功能(E/A值)。將心臟功能與BNP濃度作相關(guān)性分析,數(shù)據(jù)進(jìn)行統(tǒng)計(jì)學(xué)處理。 結(jié)果: 1.利用原核表達(dá)系統(tǒng)pET32成功構(gòu)建了pET32a.BNP重組質(zhì)粒,并制備出高純度的人Trx-BNP融合蛋白。 2.以Trx-BNP融合蛋白作抗原成功制備BNP單克隆抗體�？贵w效價(jià)高,特異性強(qiáng)。
[Abstract]:Objective: the early diagnosis and intervention of heart failure is very important in the diagnosis and treatment of heart disease. Brain natriuretic peptide (BNP), as a circulating hormone mainly derived from ventricular myocytes, is secreted rapidly and in large quantities when ventricular wall tension increases. The relationship between plasma concentration and cardiac function has been widely recognized. However, at present, the detection of BNP concentration in China depends on various kinds of different kits imported from abroad. The range and reference value of each kit are different, the results are different, lack of comparability, and the price is expensive. In this study, monoclonal antibodies against BNP were prepared for the detection of plasma BNP in normal subjects and patients with heart disease in order to evaluate the relationship between the concentration of BNP and cardiac function. It lays a foundation for the further development of domestic BNP monoclonal antibody kit. Methods: 1. The gene of BNP was spliced according to the sequence of human BNP mature protein found in GenBank. PET32a.BNP recombinant plasmid was constructed by prokaryotic expression system (pET32). Human Trx-BNP fusion protein was prepared by extraction and purification. 2. The hybridoma cell lines secreting BNP antibody were obtained by immunizing mice with Trx-BNP fusion protein. Hybridoma cells were inoculated into mouse abdominal cavity to prepare monoclonal antibodies to BNP. The antibody titers of culture supernatant and ascites of hybridoma cells were detected. The specificity of BNP antibody was detected by Western blot and immunohistochemistry. 3. The BNP monoclonal antibody was used to detect the plasma BNP concentration in normal subjects and cardiac heart disease patients by ELISA indirect method. There were 20 normal persons and 92 patients with heart disease. According to the (NYHA) cardiac function classification of the New York Heart Association, the patients were divided into three groups: cardiac function grade 鈪，
本文編號(hào)：2424854
[Abstract]:Objective: the early diagnosis and intervention of heart failure is very important in the diagnosis and treatment of heart disease. Brain natriuretic peptide (BNP), as a circulating hormone mainly derived from ventricular myocytes, is secreted rapidly and in large quantities when ventricular wall tension increases. The relationship between plasma concentration and cardiac function has been widely recognized. However, at present, the detection of BNP concentration in China depends on various kinds of different kits imported from abroad. The range and reference value of each kit are different, the results are different, lack of comparability, and the price is expensive. In this study, monoclonal antibodies against BNP were prepared for the detection of plasma BNP in normal subjects and patients with heart disease in order to evaluate the relationship between the concentration of BNP and cardiac function. It lays a foundation for the further development of domestic BNP monoclonal antibody kit. Methods: 1. The gene of BNP was spliced according to the sequence of human BNP mature protein found in GenBank. PET32a.BNP recombinant plasmid was constructed by prokaryotic expression system (pET32). Human Trx-BNP fusion protein was prepared by extraction and purification. 2. The hybridoma cell lines secreting BNP antibody were obtained by immunizing mice with Trx-BNP fusion protein. Hybridoma cells were inoculated into mouse abdominal cavity to prepare monoclonal antibodies to BNP. The antibody titers of culture supernatant and ascites of hybridoma cells were detected. The specificity of BNP antibody was detected by Western blot and immunohistochemistry. 3. The BNP monoclonal antibody was used to detect the plasma BNP concentration in normal subjects and cardiac heart disease patients by ELISA indirect method. There were 20 normal persons and 92 patients with heart disease. According to the (NYHA) cardiac function classification of the New York Heart Association, the patients were divided into three groups: cardiac function grade 鈪，
本文編號(hào)：2424854
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