抗鸡传染性支气管炎病毒单链抗体原核表达质粒的构建及其间接ELISA筛选方法的建立
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摘要
单链抗体(single-chain variable fragment, scFv)是由一条人工合成的亲水性的弹性肽链连接物将抗体的重链可变区和轻链可变区连接形成的单链蛋白,具有与天然抗体中的抗原结合位点相似的结构,保持了与天然抗体大致相似的特异性和抗原结合力。我国是一个养鸡大国,鸡的各类疾病,尤其是病毒性疾病严重危害着养鸡业的发展,降低了经济效益,甚至造成巨大的经济损失。为此,开发出免疫保护率高、安全性好、成本低廉、适于商业化生产抗病毒药物是非常重要的应对措施。单链抗体具有分子量小、便于经济有效地生产、易于基因工程改造等特点,因此比完整长度的单克隆抗体具有更大的优势和发展前景。在国内外已报道的几十种抗体片段分子中,单链抗体占35%,是目前研究较多、发展较快的重组抗体之一。然而,相较于抗人类病毒的单链抗体,抗动物病毒单链抗体的研究相对匮乏,而针对禽类病毒的单链抗体研究更是少之又少。本研究利用pOPE101-XP载体系统,构建了抗鸡传染性支气管炎病毒(IBV)单链抗体基因的原核表达质粒,并通过反应条件优化,建立了筛选单链抗体的间接酶联免疫吸附试验(ELISA),主要分为以下两部分内容:
第一部分:
直接提取免疫鸡脾脏总RNA,采用RT-PCR方法扩增抗IBV单链抗体cDNA第一链,设计特异性引物,采用PCR方法扩增出抗IBV单链抗体的VH和VL基因,利用重叠延伸PCR方法将VH和VL通过一条Linker连接成VH-Linker-VL,构建scFv。将scFv基因克隆于表达载体pOPE101-XP,构建重组质粒pOPE101-XP-scFv,双酶切鉴定。转化感受态细胞E.coli JM109(DE3),构建pOPE101-XP-scFv/JM109(DE3),菌落PCR鉴定为阳性的克隆送检测序并作序列分析。挑选阳性克隆经IPTG诱导表达,提取周质腔可溶性scFv,SDS-PAGE检验表达结果。测序结果显示成功构建抗鸡传染性支气管炎病毒单链抗体,SDS-PAGE证明可溶性单链抗体表达成功。
第二部分:
将鸡传染性支气管炎病毒M41株接种SPF鸡胚进行病毒增殖,收获的尿囊液通过透析袋浓缩后,用超速离心和蔗糖密度梯度离心方法对病毒进行纯化。SDS-PAGE和Western blotting鉴定病毒后,紫外分光光度法测病毒浓度以及待测样品中scFv表达产物的浓度。
用提纯的鸡传染性支气管炎病毒M41株作为抗原包被ELISA板,筛选与之有特异性结合的周质腔提取物中的scFv。通过反应条件的优化建立筛选抗鸡传染性支气管炎病毒单链抗体的间接ELlSA方法。本实验确定的间接ELISA各项反应参数是:包被抗原1:20稀释(61.36μg/mL),4℃包被过夜;2%脱脂奶粉37℃封闭1h;含可溶性单链抗体的待测样品1:100稀释(142.7μg/mL),37℃作用2h;MYC鼠单抗1:2000稀释,37℃作用2h;羊抗鼠抗抗体1:4000稀释,37℃作用1h。敏感性实验显示样品中的scFv浓度的最低检出量为17.83μg/mL;特异性实验显示筛选出的scFv只与IBV发生特异性结合;重复性实验的板内变异系数为2.56%,板间变异系数为2.78%,表明该筛选方法比较稳定。
Single-chain fragments variable (scFvs) contain the complete antigen binding site, which includes the variable heavy (VH) and variable light (VL) domains, of an antibody. The VH domain is linked to a VL domain by an introduced flexible polypeptide Linker. An scFv is capable of binding its target antigens with an affinity similar to that of the parent monoclonal antibody. China is a large chicken-farming country, all kinds of chicken diseases, especially viral diseases seriously affect the prospects of chicken industry, reduce the economic benefit and even cause huge economic losses. Therefore, it is of very importance to develop antiviral drugs which have ideal immunity effect, good safety and low cost. ScFvs are becoming popular therapeutic alternatives to full length monoclonal antibodies since they are smaller, most of them can be produced more economically and are easily amendable to genetic manipulation. Of the dozens of antibody fragments that have entered clinical studies at home and abroad, 35 percent are scFvs. It is one of the most rapidly developing recombinant antibodies. However, compared with human source scFvs, animal source scFvs are barely studied, and poultry scFvs research are even much rarer. This study used pOPE101-XP as vector, obtained anti-Avian Infectious Bronchitis Virus(IBV) scFvs, and established an indirect enzyme-linked immuno sorbent assay (ELISA) for anti-IBV scFv screening.
1. Construction and prokaryotic expression of anti-IBV scFv.
Chicken spleen mRNA was extracted after immunization by IBV H120. VH and VL genes were amplified by RT-PCR and PCR. ScFv gene was constructed by VH and VL chains via a Linker like VH-Linker-VL. The recombinant expression plasmid was constructed by inserting scFv into vector pOPE101-XP and was proved by sequencing. SDS-PAGE was used for analysis of the soluble scFv from periplasmic space after induced by IPTG. Sequencing result showed that anti-IBV scFv was successfully constructed. SDS-PAGE showed soluble scFv was successfully expressed.
2. Amplification and purification of IBV-M41, and establishment of an indirect ELISA for anti-IBV scFv screening.
IBV-M41 isolate was inoculated into 10-day SPF chicken embryos. The allantoic fluid (AAF) which was concentrated through dialysis bag was purified after ultracentrifugation and gradient centrifugation. The conclusion was identified by SDS-PAGE. Ultraviolet spectrophotometry was used to measure the concentration of IBV-M41. The concentration of anti-IBV scFv was detected by indirect ELISA.
Purified IBV-M41 was used to immunize mice and coat ELISA plate in order to screen the specifically binding scFvs. An indirect ELISA screening method was established by optimizing the reaction condition: IBV-M41 was diluted with 20 fold (61.36μg/mL) as the coating antibody, coating in 4℃overnight; blocking in 37℃for 1 h by 2% skim milk powder; soluble scFv was diluted with 100 fold (142.7μg/mL), reacting in 37℃for 2 h; Myc-Tag Mouse mAb was diluted with 2000 fold, reacting in 37℃for 2 h; Peroxidase-conjugated Affinipure Goat Anti-Mouse IgG was diluted with 4000 fold, reacting in 37℃for 1 h. The lower limit of detectability was 17.83μg/mL confirmed by the sensitivity test. The scFv only reacted with IBV confirmed by specificity test. Repetitive experiments (coefficient of variation was 2.56 %, 2.78 %) showed stability of the method.
第一部分:
直接提取免疫鸡脾脏总RNA,采用RT-PCR方法扩增抗IBV单链抗体cDNA第一链,设计特异性引物,采用PCR方法扩增出抗IBV单链抗体的VH和VL基因,利用重叠延伸PCR方法将VH和VL通过一条Linker连接成VH-Linker-VL,构建scFv。将scFv基因克隆于表达载体pOPE101-XP,构建重组质粒pOPE101-XP-scFv,双酶切鉴定。转化感受态细胞E.coli JM109(DE3),构建pOPE101-XP-scFv/JM109(DE3),菌落PCR鉴定为阳性的克隆送检测序并作序列分析。挑选阳性克隆经IPTG诱导表达,提取周质腔可溶性scFv,SDS-PAGE检验表达结果。测序结果显示成功构建抗鸡传染性支气管炎病毒单链抗体,SDS-PAGE证明可溶性单链抗体表达成功。
第二部分:
将鸡传染性支气管炎病毒M41株接种SPF鸡胚进行病毒增殖,收获的尿囊液通过透析袋浓缩后,用超速离心和蔗糖密度梯度离心方法对病毒进行纯化。SDS-PAGE和Western blotting鉴定病毒后,紫外分光光度法测病毒浓度以及待测样品中scFv表达产物的浓度。
用提纯的鸡传染性支气管炎病毒M41株作为抗原包被ELISA板,筛选与之有特异性结合的周质腔提取物中的scFv。通过反应条件的优化建立筛选抗鸡传染性支气管炎病毒单链抗体的间接ELlSA方法。本实验确定的间接ELISA各项反应参数是:包被抗原1:20稀释(61.36μg/mL),4℃包被过夜;2%脱脂奶粉37℃封闭1h;含可溶性单链抗体的待测样品1:100稀释(142.7μg/mL),37℃作用2h;MYC鼠单抗1:2000稀释,37℃作用2h;羊抗鼠抗抗体1:4000稀释,37℃作用1h。敏感性实验显示样品中的scFv浓度的最低检出量为17.83μg/mL;特异性实验显示筛选出的scFv只与IBV发生特异性结合;重复性实验的板内变异系数为2.56%,板间变异系数为2.78%,表明该筛选方法比较稳定。
Single-chain fragments variable (scFvs) contain the complete antigen binding site, which includes the variable heavy (VH) and variable light (VL) domains, of an antibody. The VH domain is linked to a VL domain by an introduced flexible polypeptide Linker. An scFv is capable of binding its target antigens with an affinity similar to that of the parent monoclonal antibody. China is a large chicken-farming country, all kinds of chicken diseases, especially viral diseases seriously affect the prospects of chicken industry, reduce the economic benefit and even cause huge economic losses. Therefore, it is of very importance to develop antiviral drugs which have ideal immunity effect, good safety and low cost. ScFvs are becoming popular therapeutic alternatives to full length monoclonal antibodies since they are smaller, most of them can be produced more economically and are easily amendable to genetic manipulation. Of the dozens of antibody fragments that have entered clinical studies at home and abroad, 35 percent are scFvs. It is one of the most rapidly developing recombinant antibodies. However, compared with human source scFvs, animal source scFvs are barely studied, and poultry scFvs research are even much rarer. This study used pOPE101-XP as vector, obtained anti-Avian Infectious Bronchitis Virus(IBV) scFvs, and established an indirect enzyme-linked immuno sorbent assay (ELISA) for anti-IBV scFv screening.
1. Construction and prokaryotic expression of anti-IBV scFv.
Chicken spleen mRNA was extracted after immunization by IBV H120. VH and VL genes were amplified by RT-PCR and PCR. ScFv gene was constructed by VH and VL chains via a Linker like VH-Linker-VL. The recombinant expression plasmid was constructed by inserting scFv into vector pOPE101-XP and was proved by sequencing. SDS-PAGE was used for analysis of the soluble scFv from periplasmic space after induced by IPTG. Sequencing result showed that anti-IBV scFv was successfully constructed. SDS-PAGE showed soluble scFv was successfully expressed.
2. Amplification and purification of IBV-M41, and establishment of an indirect ELISA for anti-IBV scFv screening.
IBV-M41 isolate was inoculated into 10-day SPF chicken embryos. The allantoic fluid (AAF) which was concentrated through dialysis bag was purified after ultracentrifugation and gradient centrifugation. The conclusion was identified by SDS-PAGE. Ultraviolet spectrophotometry was used to measure the concentration of IBV-M41. The concentration of anti-IBV scFv was detected by indirect ELISA.
Purified IBV-M41 was used to immunize mice and coat ELISA plate in order to screen the specifically binding scFvs. An indirect ELISA screening method was established by optimizing the reaction condition: IBV-M41 was diluted with 20 fold (61.36μg/mL) as the coating antibody, coating in 4℃overnight; blocking in 37℃for 1 h by 2% skim milk powder; soluble scFv was diluted with 100 fold (142.7μg/mL), reacting in 37℃for 2 h; Myc-Tag Mouse mAb was diluted with 2000 fold, reacting in 37℃for 2 h; Peroxidase-conjugated Affinipure Goat Anti-Mouse IgG was diluted with 4000 fold, reacting in 37℃for 1 h. The lower limit of detectability was 17.83μg/mL confirmed by the sensitivity test. The scFv only reacted with IBV confirmed by specificity test. Repetitive experiments (coefficient of variation was 2.56 %, 2.78 %) showed stability of the method.
引文
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