【摘要】：豬睪丸細(xì)胞(Swine Testis, ST)細(xì)胞系于1960年由McClurkin AW等建立,屬于成纖維細(xì)胞系,體外培養(yǎng)呈貼壁狀態(tài)。該細(xì)胞系用于豬細(xì)小病毒(PPV)、偽狂犬病毒、(PRV)、豬瘟病毒(CSFV)、豬傳染性胃腸炎病毒(TGEV)等的繁殖和豬瘟疫苗、豬細(xì)小病毒疫苗的制備,是病毒繁殖生產(chǎn)的重要細(xì)胞系之一。為了保證疫苗或生物制品的安全有效,對(duì)多種宿主細(xì)胞蛋白殘留量進(jìn)行了限定。因此,本研究對(duì)豬睪丸宿主細(xì)胞蛋白殘留特異性檢測(cè)方法進(jìn)行了初步的研究。利用本實(shí)驗(yàn)室保存的豬睪丸細(xì)胞制備了細(xì)胞抗原,采用低劑量長(zhǎng)程免疫制備多克隆抗血清。用間接ELISA和western blot方法對(duì)制備的抗血清效價(jià)和特異性進(jìn)行了測(cè)定,結(jié)果表明,抗體效價(jià)大于1：10000；瓊脂雙擴(kuò)散效價(jià)均大于1：4；制備的多克隆抗體具有良好的特異性,與DMEM、MEM、新生牛血清、胎牛血清、馬血清均沒有交叉反應(yīng)。利用純化的抗體,建立了豬睪丸宿主細(xì)胞蛋白定量檢測(cè)的間接ELISA方法。所建立的方法具有良好的重復(fù)性、特異性和靈敏度,可以檢測(cè)疫苗制品中大于25ng/mL的殘留蛋白,標(biāo)準(zhǔn)曲線的線性范圍為25-400ng/mL。對(duì)建立的間接ELISA定量檢測(cè)方法進(jìn)行了初步應(yīng)用,對(duì)市售的豬瘟疫苗中豬睪丸細(xì)胞宿主蛋白殘留量進(jìn)行了檢測(cè)。本研究建立了豬睪丸細(xì)胞蛋白殘留量的間接ELISA定量檢測(cè)方法,為生物制品中宿主細(xì)胞蛋白殘留的控制提供了可靠的檢測(cè)手段,為生物制品的安全有效提供了有力的保障。
[Abstract]:Porcine testicular (Swine Testis, ST) cell line was established by McClurkin AW et al in 1960 and belongs to fibroblast cell line. The cell line was used to reproduce porcine parvovirus (PPV), pseudorabies virus, (PRV), swine fever virus (CSFV), porcine transmissible gastroenteritis virus (TGEV) et al. The preparation of porcine parvovirus vaccine is one of the important cell lines for virus reproduction and production. In order to ensure the safety and effectiveness of vaccines or biological products, several host cell protein residues were limited. Therefore, the specific detection method of protein residues in pig testicular host cells was studied. Cell antigens were prepared from pig testicular cells preserved in our laboratory and polyclonal antisera were prepared by low dose long range immunization. The titer and specificity of the prepared antiserum were determined by indirect ELISA and western blot. The results showed that the titer of antibody was more than 1: 10000, the double diffusion titer of Agar was more than 1: 4. The polyclonal antibody prepared had good specificity. There was no cross reaction with DMEM,MEM, newborn bovine serum, fetal bovine serum and horse serum. Using purified antibody, an indirect ELISA method for quantitative detection of pig testicular host cell protein was established. The method has good reproducibility, specificity and sensitivity, and can be used to detect residual proteins larger than 25ng/mL in vaccine products. The linear range of the standard curve is 25-400ng / mL. The indirect ELISA quantitative detection method was applied to detect the residual protein of pig testicular cell host protein in swine fever vaccine. In this study, an indirect ELISA quantitative method for the detection of pig testicular protein residues was established, which provided a reliable method for the control of host cell protein residues in biological products, and provided a strong guarantee for the safety and effectiveness of biological products.
【學(xué)位授予單位】：西北民族大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S858.28

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1 王輝;張?jiān)绿m;過琴媛;賈麗麗;鐘書聲;趙玉秀;馬可;羅書榮;;疫苗制品中Vero細(xì)胞殘余蛋白含量檢測(cè)方法的建立[J];中國(guó)生物制品學(xué)雜志;2007年12期

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本文編號(hào)：2202631