CN113985030A - 快速检测猪伪狂犬抗体的免疫胶体金试纸及其制备方法 - Google Patents
快速检测猪伪狂犬抗体的免疫胶体金试纸及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种快速检测猪伪狂犬抗体的免疫胶体金试纸及其制备方法,免疫胶体金试纸在试剂条板上粘贴有NC膜、样品垫、结合垫和吸水垫,制备并纯化了金黄色葡萄球菌外膜蛋白A,并进行了胶体金标记,固定于结合垫上作为显色源;采用原核表达系统制备并纯化了PRV gE蛋白,结合于NC膜上作为捕捉抗原;采用饱和硫酸铵盐析、离子交换柱层析技术从猪血清中提纯IgG,并将纯化后的猪IgG免疫BALB/c小鼠,采用PEG1500细胞融合技术制备了鼠抗猪IgG单克隆抗体,并结合于NC膜上作为质控抗体,从而得到所述免疫胶体金试纸。本发明具有操作简单、快速、敏感和特异等特点,可用于PRV野毒感染猪的gE抗体快速检测。
Description
技术领域
本发明涉及病毒检测,具体涉及一种快速检测猪伪狂犬抗体的免疫胶体金试纸及其制备方法,涉及到分子生物学、免疫学及生物制品学,属于农业生物技术领域。
背景技术
猪伪狂犬病是由猪伪狂犬病病毒(Pseudorabies virus,PRV)引起的多种家畜、野生动物的一种以发热、脑脊髓炎为主的急性传染病。伪狂犬病(PR)一旦传入猪群,将导致巨大的经济损失,且很难根除。提高PR的诊断技术是防控该病的先决条件,只有早发现、早防控才能更加有效地降低PR的发生,避免养猪业遭受巨大的经济损失。目前诊断PRV常用的血清学方法有血清中和试验(SNT)、乳胶凝集试验(LAT)、琼脂免疫扩散试验(AGID)、血凝试验(HA)与血凝抑制试验(HI)、酶联免疫吸附试验(ELISA)等。SNT敏感性及准确性都较理想,但操作繁琐,且受技术、细胞等条件限制,不适合作为流行病学调查和大批量疫控监测使用。AGID无需特殊设备,技术操作简单,结果准确,但检测灵敏度较低;HA与HI试验操作简便,比较敏感,但受外界因素影响较大而削弱了其实用价值,乳胶凝集试验(LAT) 几分钟内即可得出结果,具有操作简单、方便、快速,但与SNT、AGID、HI方法一样无法区分野毒感染和疫苗免疫接种产生的抗体。基于重组蛋白或单克隆抗体建立的ELISA灵敏性和特异性都较高,可区分野毒感染和疫苗免疫接种产生的抗体,已开发成商品化试剂盒,但价格昂贵,检测成本较高,且操作需要专门的技术人员和分析仪器,不适合基层的使用。国内发明专利“一种检测猪伪狂犬病毒抗体的试剂盒及阻断ELISA检测方法”(专利号ZL 201110179848.1)应用针对伪狂犬病毒的单克隆抗体及阻断ELISA检测技术,该试剂盒具有较好的敏感性和特异性,但操作步骤较繁琐、需多次洗涤,易产生非特异性反应。以上方法虽然有各自的优点,但都存在操作繁琐、需要特定试验设备等限制,发明一种快速检测猪伪狂犬抗体的诊断试剂,应对疫情进行现场及时诊断是目前养猪业急需解决的问题。
免疫胶体金层析法是近年来兴起的一种快速诊断技术,其原理是将特异的抗体先固定于硝酸纤维素膜的某一区带,当该硝酸纤维素膜一端浸入样品后,由于层析作用,样品中的抗原到达固定有抗体的区带与抗体发生特异性结合,用免疫胶体金标记的抗体可显示特定颜色,从而直接判定结果。与其他检测技术相比,免疫胶体金试纸条的待检样品无需特殊处理,且用量极小(可低至50~100μL),5~10分钟即可判定结果;同时该方法不需要实验室平台和特殊仪器,没有诸如放射性同位素等有害物质污染环境,试验结果还可长期保存。近年来该技术已在兽医临床疫病检测中得到了广泛的应用。目前国内普遍用PRV gE基因缺失苗进行免疫,gE基因是PRV的非必需基因,在被野毒感染的猪血清中能检测到针对gE的抗体,这就为利用血清进行PRV野毒感染猪和gE缺失疫苗免疫接种猪进行鉴别诊断提供了条件。目前市场上出现的国内针对gE抗体诊断试纸多为非正规产品,产品质量参差不齐,鲜有国家批文,而国外类似产品成本较高(40元/样品),不适合基层推广应用。
发明内容
针对现有技术存在的上述不足,本发明的目的在于提供一种快速检测猪伪狂犬抗体的免疫胶体金试纸及其制备方法,具有操作简单、快速、敏感和特异等特点,可用于PRV野毒感染猪的gE抗体快速检测。
本发明的技术方案是这样实现的:
快速检测猪伪狂犬抗体的免疫胶体金试纸,包括试剂条板,试剂条板上粘贴有NC膜、样品垫、结合垫和吸水垫,其特征在于:所述结合垫上喷有胶体金标记的SPA;所述NC膜上分别喷有捕获抗原和质控抗体,捕获抗原由重组PRV gE蛋白构成,质控抗体为鼠抗猪IgG单克隆抗体;捕获抗原所在位置构成检测线,质控抗体所在位置构成质控线。
所述胶体金标记的SPA按如下方法制备得到,
1)SPA的分离与纯化
取改良营养肉汤培养基上生长18h的金黄色葡萄球菌培养物,采用溶菌酶裂解细胞壁释放蛋白,用PBS溶解后转入透析袋透析,透析物用小鼠IgG-sephrose 4FF亲和层析柱纯化;其中改良营养肉汤培养基包括如下质量百分比的组分:牛肉膏1%,蛋白胨3%,酵母浸膏 0.25%,葡萄糖0.1%,Na2HPO4·12H2O 0.2%,NaCl 0.3%;
2)SPA胶体金标记:用氯金酸制备胶体金颗粒,颗粒大小30nm;然后将SPA与胶体金颗粒10μg/mL进行标记,标记完后用牛血清白蛋白BSA进行封闭;标记好的金标SPA用高速离心法进行纯化,去除游离SPA和蛋白及其它小分子物质,得到最终胶体金标记的SPA。
所述重组PRV gE蛋白按如下方法制备得到,
根据PRV gE基因序列,在不改变gE蛋白氨基酸序列的情况下,根据大肠杆菌密码子偏好性优化基因序列,化学合成优化后的gE基因,并用BamHI和Xho I进行双酶切,酶切产物回收后,插入到同样经过BamHI和Xho I双酶切回收的pET28a(+)载体中,构建重组pET28a(+)/PRV-gE质粒,转化到BL21(DE3)工程菌中,采用IPTG诱导重组蛋白的表达,设置诱导温度、诱导剂浓度、诱导时间三个影响因素,筛选出最佳诱导条件大量表达重组gE蛋白;利用镍柱亲和层析法纯化诱导所得重组gE蛋白。
所述鼠抗猪IgG单克隆抗体按如下方法制备得到,
首先采用饱和硫酸铵盐析、离子交换柱层析技术从猪血清中提纯IgG;然后以纯化的IgG 作为免疫原,采用长程免疫法免疫BALB/c小鼠,取4次免疫后的脾细胞与SP2/0骨髓瘤细胞用PEG-1500进行细胞融合,将纯化的猪IgG作为抗原包被酶标孔,对融合后细胞上清进行筛选,将获得的阳性孔进行亚克隆,经过2次亚克隆,不断反复筛选得到了2株较稳定分泌抗体的杂交瘤细胞株,将获得的2株配对的杂交瘤细胞分别腹腔注射BALB/c小鼠,制备单抗腹水,先使用辛酸硫酸法初步纯化小鼠腹水,再采用Protein A柱纯化,从而得到鼠抗猪IgG单克隆抗体。
快速检测猪伪狂犬抗体的免疫胶体金试纸的制备方法,步骤如下,
1)标金结合垫的制备
1.1)SPA的分离与纯化
取改良营养肉汤培养基上生长18h的金黄色葡萄球菌培养物,采用溶菌酶裂解细胞壁释放蛋白,用PBS溶解后转入透析袋透析,透析物用小鼠IgG-sephrose 4FF亲和层析柱纯化;其中改良营养肉汤培养基包括如下质量百分比的组分:牛肉膏1%,蛋白胨3%,酵母浸膏 0.25%,葡萄糖0.1%,Na2HPO4·12H2O 0.2%,NaCl 0.3%;
1.2)SPA胶体金标记:用氯金酸制备胶体金颗粒,颗粒大小30nm;然后将SPA与胶体金颗粒10μg/mL进行标记,标记完后用牛血清白蛋白BSA进行封闭;标记好的金标SPA用高速离心法进行纯化,去除游离SPA和蛋白及其它小分子物质,得到最终胶体金标记的SPA;
1.3)将结合垫经处理液浸泡后,干燥;干燥后喷胶体金标记的SPA,然后切成7mm的宽度,即得标金结合垫;
2)样品垫的制备
将样品垫经处理液浸泡后,干燥;样品垫切成18mm宽度;
3)分别制备重组PRV gE蛋白和鼠抗猪IgG单克隆抗体
重组PRV gE蛋白的制备:根据PRV gE基因序列,在不改变gE蛋白氨基酸序列的情况下,根据大肠杆菌密码子偏好性优化基因序列,化学合成优化后的gE基因,并用BamHI和Xho I进行双酶切,酶切产物回收后,插入到同样经过BamHI和Xho I双酶切回收的pET28a(+)载体中,构建重组pET28a(+)/PRV-gE质粒,转化到BL21(DE3)工程菌中,采用 IPTG诱导重组蛋白的表达,设置诱导温度、诱导剂浓度、诱导时间三个影响因素,筛选出最佳诱导条件大量表达重组gE蛋白;利用镍柱亲和层析法纯化诱导所得重组gE蛋白,从而得到重组PRVgE蛋白;
鼠抗猪IgG单克隆抗体制备:首先采用饱和硫酸铵盐析、离子交换柱层析技术从猪血清中提纯IgG;然后以纯化的IgG作为免疫原,采用长程免疫法免疫BALB/c小鼠,取4次免疫后的脾细胞与SP2/0骨髓瘤细胞用PEG-1500进行细胞融合,将纯化的猪IgG作为抗原包被酶标孔,对融合后细胞上清进行筛选,将获得的阳性孔进行亚克隆,经过2次亚克隆,不断反复筛选得到了2株较稳定分泌抗体的杂交瘤细胞株,将获得的2株配对的杂交瘤细胞分别腹腔注射BALB/c小鼠,制备单抗腹水,先使用辛酸硫酸法初步纯化小鼠腹水,再采用ProteinA柱纯化,从而得到鼠抗猪IgG单克隆抗体;
4)试剂条板的制备:撕去背衬上的不干胶纸,分别贴上NC膜、标金结合垫、样品垫和吸水垫;在NC膜分别喷上重组PRV gE蛋白和鼠抗猪IgG单克隆抗体,线宽1mm,喷完后干燥;
5)试剂条板切条:将步骤4)制备的试剂条板切成检测需要尺寸的试剂条,立即放入有干燥剂的铝箔袋内并封口;或将试剂条放入测试卡盒内,立即放入铝箔袋内并封口。
检测原理:本发明检测时滴加待测样品后,样品溶液在毛细作用下,沿着样品垫向右移动,移动到金标垫时,样品溶液溶解胶体金标记的SPA。当猪血清样品中含有PRV抗体时,抗体IgG Fc段将与胶体金标记的SPA结合并一起向右移动,到达固定有PRV gE抗原的检测线时,PRV抗体IgG Fab段的可变区将与PRV gE抗原决定簇结合,使胶体金滞留于检测线上,检测线显示红色。样品中待测物含量越高,检测线上结合的胶体金标记抗体越多,红色颜色越深。当样品中不含有待测物时,检测线将不显红色。无论血清样品中含不含有待测物,猪血清中的IgGFc段会与胶体金标记SPA结合,过量的结合物都会与质控线的鼠抗猪IgG单抗结合滞留于质控线,形成一条蓝色线。因此若质控线未形成一条蓝色线,则说明试纸条失效,需要重新更换试纸条进行检测,即若检测线和质控线均显色线,则为阳性样品;若检测线不显红色线,质控线显蓝色线,则为阴性样品。本发明使用的是间接法检测抗体。
相比现有技术,本发明具有如下有益效果:
本发明所制备的PRV gE抗体胶体金测试卡具有操作简单、快速、敏感和特异等特点,可用于PRV野毒感染猪的gE抗体快速检测,10min左右出结果,PRV血清最低检测量为 1:1600,将在猪伪狂犬病的防控中发挥重要作用。
附图说明
图1是SPA免疫亲和层析纯化图。
图2是纯化的SPA蛋白SDS-PAGE电泳鉴定结果图。
图3是SPA与单抗Western Blot鉴定结果图。
图4是重组质粒pET28a(+)-gE的酶切鉴定结果图。图中,M:DNA分子质量标准;1:重组质粒经BamH I、Xhol I双酶切。
图5是重组质粒pET28a(+)-gE测序部分结果图。
图6是pET28a(+)-gE重组质粒转化结果图。图中左边为空的BL21(DE3);右边为转化后的结果图。
图7是pET28a(+)-gE重组蛋白纯化电泳图。图中,M:蛋白质分子质量标准;1-4:PRVgE 蛋白。
图8是重组gE蛋白的Western blot检测结果图。
图9是10%SDS-PAGE电泳结果图。
图10是SDS-PAGE电泳检测抗体纯度结果图。图中,M:蛋白分子质量标准;1:样品;2:腹水原液;3、4:纯化时穿透液;5、6:λ280nm处吸收峰洗脱液图。
图11是单抗亚型鉴定结果图。
图12是PRV gE抗体免疫胶体金测试卡阴性、阳性图。
图13是敏感性试验结果图。图中标号1-7分别按1:100、1:200、1:400、1:800、1:1600、 1:3200、1:6400稀释。
具体实施方式
本发明构思:制备并纯化了金黄色葡萄球菌外膜蛋白A(SPA),并进行了胶体金标记,固定于结合垫上作为显色源;采用原核表达系统制备并纯化了PRV gE蛋白,结合于硝酸纤维素膜上作为捕捉抗原;采用饱和硫酸铵盐析、离子交换柱层析技术从猪血清中提纯IgG,并将纯化后的猪IgG免疫BALB/c小鼠,采用PEG1500细胞融合技术制备了鼠抗猪IgG单克隆抗体,并将制备的单抗结合于硝酸纤维膜上作为质控抗体,从而制备得到检测猪伪狂犬抗体的免疫胶体金试纸。
本发明具体技术方案如下:
快速检测猪伪狂犬抗体的免疫胶体金试纸,包括试剂条板,试剂条板上粘贴有NC膜、样品垫、结合垫和吸水垫,其特征在于:所述结合垫上喷有胶体金标记的SPA;所述NC膜上分别喷有捕获抗原和质控抗体,捕获抗原由重组PRV gE蛋白构成,质控抗体为鼠抗猪IgG单克隆抗体;捕获抗原所在位置构成检测线,质控抗体所在位置构成质控线。
所述胶体金标记的SPA按如下方法制备得到,
1)SPA的分离与纯化
取改良营养肉汤培养基上生长18h的金黄色葡萄球菌培养物,采用溶菌酶裂解细胞壁释放蛋白,用PBS溶解后转入透析袋透析,透析物用小鼠IgG-sephrose 4FF亲和层析柱纯化;其中改良营养肉汤培养基包括如下质量百分比的组分:牛肉膏1%,蛋白胨3%,酵母浸膏 0.25%,葡萄糖0.1%,Na2HPO4·12H2O 0.2%,NaCl 0.3%;
2)SPA胶体金标记
用氯金酸制备胶体金颗粒,颗粒大小30nm;然后将SPA与胶体金颗粒10μg/mL进行标记,标记完后用牛血清白蛋白BSA进行封闭;标记好的金标SPA用高速离心法进行纯化,去除游离SPA和蛋白及其它小分子物质,得到最终胶体金标记的SPA。
所述重组PRV gE蛋白按如下方法制备得到,
根据PRV gE基因序列,在不改变gE蛋白氨基酸序列的情况下,根据大肠杆菌密码子偏好性优化基因序列,化学合成优化后的gE基因,并用BamHI和Xho I进行双酶切,酶切产物回收后,插入到同样经过BamHI和Xho I双酶切回收的pET28a(+)载体中,构建重组pET28a(+)/PRV-gE质粒,转化到BL21(DE3)工程菌中,采用IPTG诱导重组蛋白的表达,设置诱导温度、诱导剂浓度、诱导时间三个影响因素,筛选出最佳诱导条件大量表达重组gE蛋白;利用镍柱亲和层析法纯化诱导所得重组gE蛋白。
所述鼠抗猪IgG单克隆抗体按如下方法制备得到,
首先采用饱和硫酸铵盐析、离子交换柱层析技术从猪血清中提纯IgG;然后以纯化的IgG 作为免疫原,采用长程免疫法免疫BALB/c小鼠,取4次免疫后的脾细胞与SP2/0骨髓瘤细胞用PEG-1500进行细胞融合,将纯化的猪IgG作为抗原包被酶标孔,对融合后细胞上清进行筛选,将获得的阳性孔进行亚克隆,经过2次亚克隆,不断反复筛选得到了2株较稳定分泌抗体的杂交瘤细胞株,将获得的2株配对的杂交瘤细胞分别腹腔注射BALB/c小鼠,制备单抗腹水,先使用辛酸硫酸法初步纯化小鼠腹水,再采用Protein A柱纯化,从而得到鼠抗猪IgG单克隆抗体。
下面结合附图和具体实施方式对本发明进行详细说明。
1、金黄色葡萄球菌外膜蛋白A(SPA)的分离与纯化
为了获得纯度高、活性好的金黄色葡萄球菌蛋白A(SPA),取改良营养肉汤培养基(牛肉膏1%,蛋白胨3%,酵母浸膏0.25%,葡萄糖0.1%,Na2HPO4·12H2O 0.2%,NaCl0.3%) 上生长18h的金黄色葡萄球菌培养物,采用溶菌酶裂解细胞壁释放蛋白质,用磷酸盐缓冲溶液(0.01M,pH7.4 PBS)溶解后转入透析袋透析,透析物用小鼠IgG-sephrose 4FF亲和层析柱纯化(见图1),采用二喹啉甲酸(BCA)法测蛋白质浓度。结果表明:SPA浓度为0.933mg /mL;聚丙烯酰胺凝胶电泳(SDS-PAGE)测定SPA纯度,结果在42ku处有明显的电泳条带(见图2),蛋白免疫印迹(Western-blot)对蛋白进行特异性鉴定,进一步证实小鼠IgG与SPA在42ku处发生较强的反应(图3);以SPA为抗原,提纯的小鼠IgG为样品进行酶联免疫吸附试验(ELISA)检测,结果显示小鼠IgG稀释到1:25600时仍能与SPA结合,说明提取的SPA活性较高(见表1)。
表1 SPA与提纯的小鼠IgG的反应性
2、PRV gE蛋白的原核表达与鉴定
2.1 pET28a(+)/PRV gE重组载体的构建
为了实现PRV gE蛋白在大肠杆菌中的高效表达,本发明根据GenBank发表的PRVgE 全基因序列(GenBank登录号:MH521042.1),在不改变gE蛋白氨基酸序列(见序列1)的情况下,根据大肠杆菌密码子偏好性优化基因序列,通过化学方法合成优化后的gE基因,见序列2;并用BamH I和Xho I进行双酶切,酶切产物回收后,插入到同样经过BamH I和 Xho I双酶切回收的pET28a(+)载体中,构建重组pET28a(+)/PRV-gE质粒,将连接产物转化入大肠杆菌感受态细胞BL21(DE3)中,筛选出阳性克隆,并对阳性克隆用BamH I和 Xho I进行双酶切鉴定。将酶切鉴定正确的重组质粒送至上海生工生物工程股份有限公司进行测序鉴定。重组质粒pET28a(+)/PRV gE通过BamH I和Xho I双酶切鉴定,结果如图4所示,在1749bp(目的基因)及5000bp(空载体pET28a(+))处均出现了核酸电泳条带,此结果与预期相符合。
gE重组菌高通量测序结果如图5所示,结果表明其各连接位点以及阅读框架正确,测得的序列与预期相符合。
2.2 gE重组质粒的转化结果
重组质粒转化到BL21(DE3)感受态细胞中后,涂布到LB(Kana)平板上生长。37℃倒置培养过夜后发现,pET28a(+)-AFP/BL21(DE3)平板上布满了单菌落结果如图6所示,说明gE重组质粒成功转化到pET-28a(+)的BL21(DE3)中。
2.3重组gE蛋白的诱导表达条件筛选
将pET28a(+)-gE重组质粒转化大肠杆菌BL21(DE3)感受态细胞中,挑取单菌落接种于 5mL含卡那霉素LB液体培养基中,37℃振荡培养至OD600nm值在0.6—0.8之间,将各培养管按IPTG浓度梯度(0.1mmol/L、0.5mmol/L、1mmol/L)、温度梯度(16℃、30℃、37℃) 以及时间梯度(10,12,14,16,18,20小时)进行诱导筛选,所有管按设定的诱导时间取样一次(每次0.5mL),将样品进行SDS-PAGE电泳分析确定最佳诱导条件。从IPTG诱导浓度、诱导温度以及诱导时间三个因素进行重组gE蛋白诱导条件的筛选,最终确定 pET28a(+)-gE/BL21(DE3)重组菌株在诱导条件为37℃,IPTG浓度为0.1mmol/L诱导6h时,重组gE蛋白表达最高。
2.4 gE蛋白的大量表达、纯化与鉴定
按2.3步骤优化得到的最佳条件大规模进行诱导表达重组gE蛋白,采用His-tag镍柱对重组gE蛋白(51KD)进行纯化。样品上柱前,先用亲和平衡液平衡层析柱,然后将所得样品过柱,收集少量穿透液。用亲和平衡液过柱,除去样品中的杂蛋白。再用不同浓度梯度咪唑的亲和洗脱液洗脱镍柱,收集得到纯化的重组gE蛋白。经12%SDS-PAGE分析纯化结果,表明纯化后的gE蛋白纯度达到了90%以上,如图7所示。纯化后的重组蛋白经Western-blot鉴定,在51KD处出现特异性条带,与预期的目的蛋白的大小相符合,见图8。
3鼠抗猪IgG单克隆抗体的制备
3.1猪IgG的分离纯化
血清中IgG的粗提,采用常规的饱和硫酸铵盐析法;血清IgG的分离纯化及鉴定,按常规方法处理DEAE52,装柱平衡后,将IgG粗提液加入DEAE52柱,用0.01MpH7.4 PBS(NaCl0.03M)洗脱,流速为30-40滴/分钟,分管收集,用10%磺基水杨酸测蛋白或采用紫外检测仪按蛋白峰收集,最后用10﹪SDS—PAGE电泳鉴定其纯度。图9中,1、2为第一次提纯的 IgG;3、4为第二次提纯的IgG。M为标准分子量蛋白质。可见第一次提纯的IgG达到了电泳纯,已将IgG从样品中分离出来。紫外分光光度计测得IgG的浓度为2.24mg/ml。
3.2鼠抗猪IgG单克隆抗体杂交瘤细胞株的建立
将上述纯化的猪IgG作为免疫原免疫BALB/c小鼠,采用长程免疫法免疫BALB/c小鼠,取4次免疫后的脾细胞与SP2/0骨髓瘤细胞用PEG 1500进行了一次细胞融合,细胞融合率为 90.36%。建立间接ELISA,用于检测杂交瘤细胞上清中的单克隆抗体,初检阳性率分别为 13.83%。选择阳性值较高的30个孔亚克隆,经过2次亚克隆,不断反复筛选得到了2株较稳定分泌抗体的杂交瘤细胞株,命名为6B4、4B8。细胞融合的筛选结果如表2所示。将获得的2株配对的杂交瘤细胞6B4、4B8分别腹腔注射BALB/c小鼠,收集小鼠腹水,先采用辛酸-硫酸铵法初步纯化小鼠腹水,再采用ProteinA柱进一步分离纯化,纯化后的抗体进行 SDS-PAGE电泳鉴定,如图10所示。ELISA证实所获得的单抗是特异性针对IgG,如表3、表4;6B4、4B8经反复冻存,复苏及多次传代,仍能分泌高效价抗体,分泌抗体均属IgG1、κ型,如图11所示。
表2细胞融合及阳性率一览表
表3 4B8杂交瘤细胞株上清与不同Ig反应性(x±s,n=3)
*阳性血清:指抗SIgA、IgM、IgG的小鼠血清,分别用于相应抗原包被孔,作为阳性对照
表4 6B4杂交瘤细胞上清与不同Ig反应性(x±s,n=3)
*阳性血清:指抗SIgA、IgM、IgG的小鼠血清,分别用于相应抗原包被孔,作为阳性对照
3.3、单抗的生物特性鉴定
3.3.1阳性杂交瘤细胞稳定性试验
4B8、6B4细胞株连续继代培养6个月以上,其培养上清效价仍保持在1:128-1:256。这些细胞株冻存1个月,2个月,4个月,8个月和12个月后复苏,其诱生腹水的Mabs效价仍保持在25600-51200之间。
3.3.2杂交瘤细胞染色体分析
4B8、6B4两株杂交瘤细胞分裂相染色体形态观察,均可见到端着丝点和中间着丝点,染色体数目介于100—110条之间,具有杂交瘤细胞染色体特征。
3.3.3单抗的亚型鉴定
将4B8、6B4无血清培养上清分别用0.01mol/LPBS(pH7.4)1:16稀释,使得抗体浓度在0.1-1ug/ml之间;按Mouse Monoclonal Antibody Isotyping Kit操作手册进行。结果显示在试纸条的G1,κ区各出现一条清晰的条带,如图11,表明单抗的亚型属IgG1,κ型。
3.3.4单克隆抗体的特异性分析
分别将提纯的猪的IgG、SIgA、IgM以相同的浓度(4ug/ml)包被酶标条来检测6B4、4B8 细胞上清特异性。从表所得的数据可得出:已获得的抗猪IgG单抗与IgM、SIgA间接ELISA 反应均为阴性;而与IgG反应细胞上清效价高于1:128。
4、猪伪狂犬抗体免疫胶体金层析法建立
4.1 SPA的胶体金标记及纯化
用氯金酸制备胶体金颗粒,颗粒大小30nm。取胶体金溶液,用0.1mol/L K2CO3调pH至 6.2,在磁力搅拌器的作用下缓慢逐滴加入适量的1g/L SPA,使其终浓度为40ug/mL,继续搅拌20min,室温放置30min。缓慢加入适量的3%PEG20000,使其终浓度为0.08%,继续搅拌20min,室温放置30min。然后将该溶液4000r/min 4℃离心20min,吸出上清,弃黑色沉淀;再将上清12000r/min 4℃离心1h;弃上清留沉淀,沉淀用稀释液恢复至原体积的1/10,即为纯化后的金标SPA。
4.2样品垫和结合垫的特殊处理
样品垫经处理液浸泡后,在37℃的温度下干燥2h后,切成18mm宽度,用于过滤样品中的有形成分。结合垫处理方式与样品垫相同,均需用缓冲液浸泡,干燥后喷金标抗体,置于样品垫下,并与硝酸纤维素膜(nitrocellulose filter membrane,简称NC膜)相连。
4.3试剂条的生产工艺流程的建立
4.3.1金标SPA的制备:用氯金酸制备胶体金颗粒,颗粒大小30nm;然后将SPA与胶体金颗粒10μg/mL进行标记,标记完后用牛血清白蛋白(BSA)进行封闭。标记好的金标SPA用高速离心法进行纯化,去除游离SPA和蛋白及其它小分子物质。
4.3.2结合垫和样品垫的处理:将结合垫和样品垫放入相应的缓冲液中(见表5、表6) 浸泡、干燥,结合垫喷标记好的金标抗体,然后干燥。将结合垫切成7mm的宽度,样品垫切成18mm宽度。
表5结合垫处理液设计表
表6样品垫处理液设计
4.3.3试剂条板的制备:撕去背衬上的不干胶纸,分别贴上NC膜、结合垫(已喷有金标 SPA)、样品垫和吸水垫纸。
4.3.4 NC膜的喷样:将制备好的试剂条板上的NC膜分别喷上捕获抗原(重组PRVgE蛋白)和质控抗体(鼠抗猪IgG单克隆抗体),线宽1mm,喷完后在37℃的温度下干燥。
4.3.5试剂条板切条和测试卡的组装:将NC膜喷样处理的试剂条板切成50mm×10mm 的试剂条,立即放入有干燥剂的铝箔袋内,封口;或将试剂条板切成30mm×10mm的试剂条放入测试卡盒内,立即放入铝箔袋内封口。测试卡阴、阳性测试图如图12所示,左边为阳性样品;右边为阴性样品。
5、胶体金试纸敏感性和特异性分析
5.1猪伪狂犬抗体胶体金试纸条的敏感性试验
将猪伪狂犬gE阳性血清用PBS(0.01M,pH7.4)按1:100、1:200、1:400、1:800、1:1600、 1:3200、1:6400稀释,使用本发明研制的诊断试纸进行敏感性测试。结果显示PRV阳性血清最低检测量为1:1600。见图13所示。
5.2猪伪狂犬抗体胶体金试纸条的特异性试验
使用猪圆环病毒、猪繁殖与呼吸综合征病毒、猪细小病毒及猪伪狂犬gE抗体血清进行特异性试验,猪伪狂犬gE抗体血清反应为阳性,猪圆环病毒、猪繁殖与呼吸综合征病毒、猪细小病毒血清为阴性,重复三次,结果一致。说明本试纸与常见的猪病其他血清无交叉反应和假阳性反应,特异性较好。
5.3猪伪狂犬抗体胶体金试纸的稳定性试验
取3个批次的猪伪狂犬抗体免疫胶体金试纸(每批次30条),于室温条件下(25℃)条件下保存,在第4月、6月、8月、10月和12月分别从每个批次取出15条试纸,每个批次试纸条分别检测8份阳性样品和7份阴性样品,用于检测试纸条的稳定性。在第4月、6月和8月的试验中,所有检测结果均与预期结果一致;在第10月的试验中,有两个批次的试纸条各出现1份阳性样品的检测结果为阴性,出现检测结果与预期不相符。试验结果表明该试纸条在常温条件下的有效保存期至少为8个月。
5.4猪伪狂犬抗体胶体金试纸的评价
取经美国IDEXX PRV gE抗体ELISA试剂盒检测的样品125份,已确定其中阳性样品52 份,阴性样品73份。经自制的PRV gE抗体胶体金诊断试剂检测,检测阳性率、阴性率与标准试剂盒进行比较,对自制的PRV gE抗体胶体金进行质量评价。结果显示:经本方法PRV 抗体胶体金测试卡检测,发现检测的灵敏度为96.2%,特异性97.3%,如表7所示,表明研制的胶体金测试卡具有较高的灵敏度和特异性、能满足临床快速检测要求,具有潜在推广应用价值。
表7 PRV抗体胶体金测试卡评价
最后需要说明的是,本发明的上述实施例仅仅是为说明本发明所作的举例,而并非是对本发明的实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其他不同形式的变化和变动。这里无法对所有的实施方式予以穷举。凡是属于本发明的技术方案所引申出的显而易见的变化或变动仍处于本发明的保护范围之列。
SEQUENCE LISTING
<110> 重庆市动物疫病预防控制中心;重庆理工大学
<120> 快速检测猪伪狂犬抗体的免疫胶体金试纸及其制备方法
<130> 1
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 579
<212> PRT
<213> 人工合成
<400> 1
Met Arg Pro Phe Leu Leu Arg Ala Ala Gln Leu Leu Ala Leu Leu Ala
1 5 10 15
Leu Ala Leu Ser Thr Glu Ala Pro Ser Leu Ser Ala Glu Thr Thr Pro
20 25 30
Gly Pro Val Thr Glu Val Pro Ser Pro Ser Ala Glu Val Trp Asp Asp
35 40 45
Leu Ser Thr Glu Ala Asp Asp Asp Asp Leu Asn Gly Asp Leu Asp Gly
50 55 60
Asp Asp Arg Arg Ala Gly Phe Gly Ser Ala Leu Ala Ser Leu Arg Glu
65 70 75 80
Ala Pro Pro Ala His Leu Val Asn Val Ser Glu Gly Ala Asn Phe Thr
85 90 95
Leu Asp Ala Arg Gly Asp Gly Ala Val Leu Ala Gly Ile Trp Thr Phe
100 105 110
Leu Pro Val Arg Gly Cys Asp Ala Val Ser Val Thr Thr Val Cys Phe
115 120 125
Glu Thr Ala Cys His Pro Asp Leu Val Leu Gly Arg Ala Cys Val Pro
130 135 140
Glu Ala Pro Glu Met Gly Ile Gly Asp Tyr Leu Pro Pro Glu Val Pro
145 150 155 160
Arg Leu Arg Arg Glu Pro Pro Ile Val Thr Pro Glu Arg Trp Ser Pro
165 170 175
His Leu Ser Val Leu Arg Ala Thr Pro Asn Asp Thr Gly Leu Tyr Thr
180 185 190
Leu His Asp Ala Ser Gly Pro Arg Ala Val Phe Phe Val Ala Val Gly
195 200 205
Asp Arg Pro Pro Ala Pro Ala Asp Pro Val Gly Pro Ala Arg His Glu
210 215 220
Pro Arg Phe His Ala Leu Gly Phe His Ser Gln Leu Phe Ser Pro Gly
225 230 235 240
Asp Thr Phe Asp Leu Met Pro Arg Val Val Ser Asp Met Gly Asp Ser
245 250 255
Arg Glu Asn Phe Thr Ala Thr Leu Asp Trp Tyr Tyr Ala Arg Ala Pro
260 265 270
Pro Arg Cys Leu Leu Tyr Tyr Val Tyr Glu Pro Cys Ile Tyr His Pro
275 280 285
Arg Ala Pro Glu Cys Leu Arg Pro Val Asp Pro Ala Cys Ser Phe Thr
290 295 300
Ser Pro Ala Arg Ala Arg Leu Val Ala Arg Arg Ala Tyr Ala Ser Cys
305 310 315 320
Ser Pro Leu Leu Gly Asp Arg Trp Leu Thr Ala Cys Pro Phe Asp Ala
325 330 335
Phe Gly Glu Glu Val His Thr Asn Ala Thr Ala Asp Glu Ser Gly Leu
340 345 350
Tyr Val Leu Val Met Thr His Asn Gly His Val Ala Thr Trp Asp Tyr
355 360 365
Thr Leu Val Ala Thr Ala Ala Glu Tyr Val Thr Val Ile Lys Glu Leu
370 375 380
Thr Ala Pro Ala Arg Ala Pro Gly Thr Pro Trp Gly Pro Gly Gly Gly
385 390 395 400
Asp Asp Ala Ile Tyr Val Asp Gly Val Thr Thr Pro Ala Pro Pro Ala
405 410 415
Arg Pro Trp Asn Pro Tyr Gly Arg Thr Thr Pro Gly Arg Leu Phe Val
420 425 430
Leu Ala Leu Gly Ser Phe Val Met Thr Cys Val Val Gly Gly Ala Ile
435 440 445
Trp Leu Cys Val Leu Cys Ser Arg Arg Arg Ala Ala Ser Arg Pro Phe
450 455 460
Arg Val Pro Thr Arg Ala Arg Thr His Met Leu Ser Pro Val Tyr Thr
465 470 475 480
Ser Leu Pro Thr His Glu Asp Tyr Tyr Asp Gly Asp Asp Asp Asp Asp
485 490 495
Glu Glu Ala Gly Val Ile Arg Arg Arg Pro Ala Ser Pro Gly Gly Asp
500 505 510
Ser Gly Tyr Glu Gly Pro Tyr Ala Ser Leu Asp Pro Glu Asp Glu Phe
515 520 525
Ser Ser Asp Glu Asp Asp Gly Leu Tyr Val Arg Pro Glu Glu Ala Pro
530 535 540
Arg Ser Gly Phe Asp Val Trp Phe Arg Asp Pro Glu Lys Pro Glu Val
545 550 555 560
Thr Asn Gly Pro Asn Tyr Gly Val Thr Ala Asn Arg Leu Leu Met Ser
565 570 575
Arg Pro Ala
<210> 2
<211> 1740
<212> DNA
<213> 人工合成
<400> 2
atgcggccct ttctgctgcg cgccgcgcag ctcctggcgc tgctggccct ggcgctctcc 60
accgaggccc cgagcctctc cgccgagacg accccgggcc ccgtcaccga ggtcccgagt 120
ccctcggccg aggtctggga cgacctctcc accgaggccg acgacgatga cctcaacggc 180
gacctcgacg gcgacgaccg ccgcgcgggc ttcggctcgg ccctcgcatc cctgagggag 240
gcgcccccgg cccatctggt gaacgtgtcc gagggcgcca acttcaccct cgacgcgcgc 300
ggcgacggcg ccgtgctggc cgggatctgg acgttcctgc ccgtccgcgg ctgcgacgcc 360
gtgtcggtga ccacggtgtg cttcgagacc gcgtgccacc cggacctggt gctgggccgc 420
gcctgcgtcc ccgaggcccc ggagatgggc atcggcgact acctgccgcc cgaggtgccg 480
cggctccggc gcgagccgcc catcgtcacc ccggagcggt ggtcgccgca cctgagcgtc 540
ctgcgggcca cgcccaacga cacgggcctc tacacgctgc acgacgcctc ggggccgcgg 600
gccgtgttct ttgtggcggt gggcgaccgg ccgcccgcgc cggcggaccc ggtgggcccc 660
gcgcgccacg agccccgctt ccacgcgctc ggcttccact cgcagctctt ctcgcccggg 720
gacacgttcg acctgatgcc gcgcgtggtc tcggacatgg gcgactcgcg cgagaacttt 780
accgccacgc tggactggta ctacgcgcgc gcgcccccgc ggtgcctgct gtactacgtg 840
tacgagccct gcatctacca cccgcgcgcg cccgagtgcc tgcgcccggt ggacccggcg 900
tgcagcttca cctcgccggc gcgcgcgcgg ctggtggcgc gccgcgcgta cgcctcgtgc 960
agcccgctgc tcggggaccg gtggctgacc gcctgcccct tcgacgcctt cggcgaggag 1020
gtgcacacga acgccaccgc ggacgagtcg gggctgtacg tgctcgtgat gacccacaac 1080
ggccacgtcg ccacctggga ctacacgctc gtcgccaccg cggccgagta cgtcacggtc 1140
atcaaggagc tgacggcccc ggcccgggcc ccgggcaccc cgtggggccc cggcggcggc 1200
gacgacgcga tctacgtgga cggcgtcacg acgccggcgc cgcccgcgcg cccgtggaac 1260
ccgtacggcc ggacgacgcc cgggcggctg tttgtgctgg cgctgggctc cttcgtgatg 1320
acgtgcgtcg tcgggggggc catctggctc tgcgtgctgt gctcccggcg ccgggcggcc 1380
tcgcggccgt tccgggtgcc gacgcgggcg cggacgcaca tgctctctcc ggtgtacacc 1440
agcctgccca cgcacgagga ctactacgac ggcgacgacg acgacgacga ggaggcgggc 1500
gtcatccgcc ggcggcccgc ctcccccggc ggagacagcg gctacgaggg gccgtacgcg 1560
agcctggacc ccgaggacga gttcagcagc gacgaggacg acgggctgta cgtgcgcccc 1620
gaggaggcgc cccgctccgg cttcgacgtc tggttccgcg atccggagaa accggaagtg 1680
acgaatggac ccaactatgg cgtgaccgcc aaccgcctgt tgatgtcccg ccccgcttaa 1740
Claims (5)
1.快速检测猪伪狂犬抗体的免疫胶体金试纸,包括试剂条板,试剂条板上粘贴有NC膜、样品垫、结合垫和吸水垫,其特征在于:所述结合垫上喷有胶体金标记的SPA;所述NC膜上分别喷有捕获抗原和质控抗体,捕获抗原由重组PRV gE蛋白构成,质控抗体为鼠抗猪IgG单克隆抗体;捕获抗原所在位置构成检测线,质控抗体所在位置构成质控线。
2.根据权利要求1所述的快速检测猪伪狂犬抗体的免疫胶体金试纸,其特征在于:所述胶体金标记的SPA按如下方法制备得到,
1)SPA的分离与纯化
取改良营养肉汤培养基上生长18 h的金黄色葡萄球菌培养物,采用溶菌酶裂解细胞壁释放蛋白,用PBS溶解后转入透析袋透析,透析物用小鼠IgG-sephrose 4FF亲和层析柱纯化;其中改良营养肉汤培养基包括如下质量百分比的组分:牛肉膏1%,蛋白胨3%,酵母浸膏0.25%,葡萄糖0.1%,Na2HPO4·12H2O 0.2%,NaCl 0.3%;
SPA胶体金标记:用氯金酸制备胶体金颗粒,颗粒大小30nm;然后将SPA与胶体金颗粒10µg/mL进行标记,标记完后用牛血清白蛋白BSA进行封闭;标记好的金标SPA用高速离心法进行纯化,去除游离SPA和蛋白及其它小分子物质,得到最终胶体金标记的SPA。
3.根据权利要求1所述的快速检测猪伪狂犬抗体的免疫胶体金试纸,其特征在于:所述重组PRV gE蛋白按如下方法制备得到,
根据PRV gE基因序列,在不改变gE蛋白氨基酸序列的情况下,根据大肠杆菌密码子偏好性优化基因序列,化学合成优化后的gE基因,并用BamH I和Xho I进行双酶切,酶切产物回收后,插入到同样经过BamH I和Xho I双酶切回收的pET28a(+)载体中,构建重组 pET28a(+)/PRV-gE质粒,转化到BL21(DE3)工程菌中,采用IPTG诱导重组蛋白的表达,设置诱导温度、诱导剂浓度、诱导时间三个影响因素,筛选出最佳诱导条件大量表达重组gE蛋白;利用镍柱亲和层析法纯化诱导所得重组gE蛋白。
4.根据权利要求1所述的快速检测猪伪狂犬抗体的免疫胶体金试纸,其特征在于:所述鼠抗猪IgG单克隆抗体按如下方法制备得到,
首先采用饱和硫酸铵盐析、离子交换柱层析技术从猪血清中提纯IgG;然后以纯化的IgG作为免疫原,采用长程免疫法免疫BALB/c小鼠,取4次免疫后的脾细胞与SP2/0骨髓瘤细胞用PEG-1500进行细胞融合,将纯化的猪IgG作为抗原包被酶标孔,对融合后细胞上清进行筛选,将获得的阳性孔进行亚克隆,经过2次亚克隆,不断反复筛选得到了2株较稳定分泌抗体的杂交瘤细胞株,将获得的2株配对的杂交瘤细胞分别腹腔注射BALB/c小鼠,制备单抗腹水,先使用辛酸硫酸法初步纯化小鼠腹水,再采用Protein A柱纯化,从而得到鼠抗猪IgG单克隆抗体。
5.权利要求1所述快速检测猪伪狂犬抗体的免疫胶体金试纸的制备方法,其特征在于:步骤如下,
1)标金结合垫的制备
1.1)SPA的分离与纯化
取改良营养肉汤培养基上生长18 h的金黄色葡萄球菌培养物,采用溶菌酶裂解细胞壁释放蛋白,用PBS溶解后转入透析袋透析,透析物用小鼠IgG-sephrose 4FF亲和层析柱纯化;其中改良营养肉汤培养基包括如下质量百分比的组分:牛肉膏1%,蛋白胨3%,酵母浸膏0.25%,葡萄糖0.1%,Na2HPO4·12H2O 0.2%,NaCl 0.3%;
1.2)SPA胶体金标记:用氯金酸制备胶体金颗粒,颗粒大小30nm;然后将SPA与胶体金颗粒10µg/mL进行标记,标记完后用牛血清白蛋白BSA进行封闭;标记好的金标SPA用高速离心法进行纯化,去除游离SPA和蛋白及其它小分子物质,得到最终胶体金标记的SPA;
1.3)将结合垫经处理液浸泡后,干燥;干燥后喷胶体金标记的SPA,然后切成7mm的宽度,即得标金结合垫;
2)样品垫的制备
将样品垫经处理液浸泡后,干燥;样品垫切成18mm宽度;
3)分别制备重组PRV gE蛋白和鼠抗猪IgG单克隆抗体
重组PRV gE蛋白的制备:根据PRV gE基因序列,在不改变gE蛋白氨基酸序列的情况下,根据大肠杆菌密码子偏好性优化基因序列,化学合成优化后的gE基因,并用BamH I和Xho I进行双酶切,酶切产物回收后,插入到同样经过BamH I和Xho I双酶切回收的pET28a(+)载体中,构建重组 pET28a(+)/PRV-gE质粒,转化到BL21(DE3)工程菌中,采用IPTG诱导重组蛋白的表达,设置诱导温度、诱导剂浓度、诱导时间三个影响因素,筛选出最佳诱导条件大量表达重组gE蛋白;利用镍柱亲和层析法纯化诱导所得重组gE蛋白,从而得到重组PRV gE蛋白;
鼠抗猪IgG单克隆抗体制备:首先采用饱和硫酸铵盐析、离子交换柱层析技术从猪血清中提纯IgG;然后以纯化的IgG作为免疫原,采用长程免疫法免疫BALB/c小鼠,取4次免疫后的脾细胞与SP2/0骨髓瘤细胞用PEG-1500进行细胞融合,将纯化的猪IgG作为抗原包被酶标孔,对融合后细胞上清进行筛选,将获得的阳性孔进行亚克隆,经过2次亚克隆,不断反复筛选得到了2株较稳定分泌抗体的杂交瘤细胞株,将获得的2株配对的杂交瘤细胞分别腹腔注射BALB/c小鼠,制备单抗腹水,先使用辛酸硫酸法初步纯化小鼠腹水,再采用Protein A柱纯化,从而得到鼠抗猪IgG单克隆抗体;
4)试剂条板的制备:撕去背衬上的不干胶纸,分别贴上NC膜、标金结合垫、样品垫和吸水垫;在NC膜分别喷上重组PRV gE蛋白和鼠抗猪IgG单克隆抗体,线宽1 mm,喷完后干燥;
5)试剂条板切条:将步骤4)制备的试剂条板切成检测需要尺寸的试剂条,立即放入有干燥剂的铝箔袋内并封口;或将试剂条放入测试卡盒内,立即放入铝箔袋内并封口。
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