CN110615846B - 一种兼有IgG结合活性及生物素结合活性的双功能蛋白及其ELISA试剂盒 - Google Patents
一种兼有IgG结合活性及生物素结合活性的双功能蛋白及其ELISA试剂盒 Download PDFInfo
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Abstract
本发明属于基因工程领域。具体而言,本发明提供一种兼有IgG结合活性及生物素结合活性的双功能蛋白及其ELISA试剂盒,尤其涉及密码子优化的一种兼有IgG结合活性及生物素结合活性的双功能蛋白G‑SA,包括链球菌蛋白G(SPG)片段和SA蛋白。本发明提供的ELISA试剂盒的特异性、敏感性和稳定性较好,具有良好的应用前景。
Description
技术领域
本发明涉及免疫检测领域,特别涉及一种兼有IgG 结合活性及生物素结合活性的双功能蛋白G-SA及其ELISA试剂盒。
背景技术
目前,感染人类的吸虫有三种:即分布于亚洲的日本血吸虫,非洲中部及拉丁美洲的曼式血吸虫和非洲北部的埃及血吸虫,在中国仅日本血吸虫流行,分布于我国长江流域及南方12省,市,自治区,多为我国很重要的农业生产基地,日本血吸虫病是一种分布广泛、危害严重的人兽共患寄生虫病,对人民的健康及国家发展危害严重。
血吸虫病诊断技术主要包括病原学诊断和免疫学诊断两种,病原学诊断技术以粪便等样本中检查虫卵或孵化毛蚴为手段,但该法检出率较低,对轻度感染或早期感染的检测漏检率极高,不利于疾病的早期诊断。免疫学诊断技术检测主要包括抗原和血清抗体,相比病原学诊断技术具有简便,高效,依从性高等优势,日益受到重视并在业界研发下发展迅速。
链球菌蛋白G是一种能够和多种哺乳动物的IgG以及人血清白蛋白(HSA)相结合的蛋白质,SPG同IgG的亲和力强,结合谱广。研究表明,SPG的三个同源的氨基酸序列C1、C2和C3区域与抗体IgGFc端的结合相关,并且C3区与抗体IgG的结合能力相当于C1区的7倍(Kobatake,1990)。而SPG的A、B区则具有与抗体Fab段结合以及与血清白蛋白结合的活性,被认为会起到干扰抗体与抗原的作用以及带来非特异性反应的问题。
链霉亲和素(streptavidin,SA)是由链霉菌Streptomycesavidinii分泌的一种四聚体蛋白,与亲和素(avidin,AV)有相似生物学特性。SA可高度特异性地与生物素结合,一分子链霉亲和素可以与四分子生物素结合,形成生物素-链霉亲和素系统。该系统被认为是最稳定的非共价相互作用之一,并在生物技术领域具有广泛的应用,如分子标记、分子定位和靶向药物递送。
酶联免疫吸附试验是免疫学诊断技术的一种,该方法具有高度特异性,敏感性,检测结果与粪检阳性符合率达95%-99%。ELISA技术飞速发展,出现了诸多衍生试验,如快速ELISA,微波ELISA,Dot-ELISA等均具有良好的特异性和敏感性。
BSA系统作为ELISA法的一种,可显著提高标记免疫检测的灵敏度,在免疫学领域应用广泛。但是常规的BSA法同样不可避免的用到酶标的二抗,根据不同物种来源的一抗,二抗也需要做相应的调整,增加了实验成本也给实验结果准确性带来影响。
发明内容
本发明主要解决的技术问题是提供一种快速、高灵敏的免疫检测产品及方法,实现高效、高密度的免疫检测。
为了实现上述技术方案,本发明提供一种融合蛋白,基于SPG C3区和SA的特点,将这两段基因连接起来,重构一种兼有IgG 结合活性及生物素结合活性的双功能蛋白G-SA。
为了实现本发明的目的,本发明采用如下技术方案:
一种兼有IgG 结合活性及生物素结合活性的双功能蛋白G-SA,包括链球菌蛋白G(SPG)片段和SA蛋白,所述链球菌蛋白G(SPG)片段为含有SPG的C3区段的序列;
其中,链球菌蛋白G(SPG)片段和SA蛋白之间的连接序列为如SEQ ID NO.8所示
所述链球菌蛋白G(SPG)片段的核苷酸序列如SEQ ID NO.6所示;
SA蛋白的核苷酸序列如SEQ ID NO.7所示;
所述双功能蛋白G-SA的核苷酸序列如SEQ ID NO.5所示;
本发明同时提供一种密码子优化的序列,对SPG和SA序列进行密码子优化,拼接后蛋白表达量提高,可溶性增加。
本发明同时提供一种双功能蛋白G-SA的构建方法,所述方法为:根据G蛋白的C3片段的基因序列,从含有C3片段的菌液中PCR扩增出C3序列;
(1)通过PCR从含有SA序列的菌液中扩增出SA序列;
(2)先将扩增出的SA序列双酶切后,连接到表达载体上,经鉴定后再将C3片段双酶切后连接上去,构建重组蛋白的原核表达质粒;
(3)将所述表达载体、共表达载体转入表达宿主中培养,活化至对数生长期后加入诱导表白蛋白;
(4) 经破碎、纯化后制得融合蛋白G-SA
其中,步骤(1)中使用的引物对如SEQ ID NO.1、2所示;
步骤(2)中使用的引物对如SEQ ID NO.3、4所示;
上述方法中,融合蛋白基因的表达、纯化可采用本领域常规用于蛋白表达纯化的方法,例如将融合蛋白基因克隆入表达载体,将表达载体和/或共表达载体转入表达宿主中培养,活化至对数生长期后加入诱导表白蛋白,经破碎、纯化后得到融合蛋白。其中,本发明对表达载体、表达宿主的种类和类别不作限定,可选用本领域常规用于遗传修饰的载体和宿主,具体的,表达载体可为pET-28、pET-32、pET-15或pET-11的等,;表达宿主可选自大肠杆菌、枯草芽孢杆菌、巨大芽孢杆菌、棒状杆菌、酿酒酵母、毕赤酵母或哺乳动物细胞。
本发明中,克隆可通过例如链式酶聚合反应(PCR)完成。
进一步的,本发明提供一种ELISA试剂盒,其中所述ELISA试剂盒,所述ELISA试剂盒中包括:双功能蛋白G-SA、封闭液(5%脱脂奶粉),酶标二抗(HRP标记的生物素和亲和素),PBST,显色液(TMB底物液)、终止液(2 mol/L硫酸)。
同时,本发明提供一种ELISA试剂盒的非诊断目的的应用,所述应用为抗体检测、抗体筛选、抗原检测、病原检测、蛋白检测、蛋白相互作用筛查、高通量靶标蛋白检测、蛋白-核酸相互作用分析、药物筛选等。
附图说明
图1 SPG的结构示意图。
图2 对重组蛋白的原核表达质粒,进行PCR鉴定,其中,M:核酸标志物;1:G-SA序列;2:C3序列;3.SA序列。
图3重组质粒的双酶切鉴定,其中,M:核酸标志物1:PET-28a空载体;2:G-SA基因。
图4 SDS-PAGE分析pET-28a(+)-C3-SA表达情况,其中,M:蛋白标志物;0:无IPTG诱导;1、2、4、6、8:IPTG诱导1h,2h,4h,6h, 8h。
图5 SDS-PAGE分析pET-28a(+)-C3-SA上清蛋白纯化情况
M:蛋白标志物;1:超声破碎上清液;2:上样后;3:超声破碎沉淀;4:BindingBuffer纯化后;5:Washing Buffer纯化后;6:Strip Buffer纯化后。
图6 Western blotting检测G-SA蛋白与不同物种IgG、生物素以及His抗体的结合活性
图A:1:蛋白maker;2:鼠抗兔; 图B:1:蛋白maker;2:驴抗羊;
图C:1:蛋白maker;2:羊抗鼠; 图D:1:蛋白maker;2:兔抗山羊;
图E:1:蛋白maker;2:HRP-bio; 图F:1:蛋白maker;2:His抗体。
图7 G-SA与不同物种抗体、生物素的亲和常数曲线
图8 新型ELISA系统模式图
图9 重组蛋白最适孵育浓度摸索
图10 新型ELISA检测法的多物种检测,灵敏度检测
图11新型ELISA检测法的多物种检测,特异性检测。
具体实施方式
以下的实施例便于更好地理解本发明,但并不限定本发明。下述实施例中的实验方法,如无特殊说明,均为常规方法。下述实施例中所用的试验材料,如无特殊说明, 均为自常规生化试剂商店购买得到的。
实施例1重组蛋白的构建
1.1 生物材料
大肠埃希氏杆菌BL21购自南京诺唯赞生物科技有限公司;含SA、SPG质粒、质粒pET-28a(+)为实验室保存。
SA对应文献:[1]訾静,张月娟,万一.核心链霉亲和素在毕赤酵母中的表达及纯化[J].中国生物制品学杂志,2018,31(05):485-488.
SPG对应文献:[1]许瑞,赵登云,洪炀,陆珂,李浩,林矫矫,冯金涛,徐玉梅,朱传刚.链球菌蛋白G的结构域重构、表达及鉴定[J].中国动物传染病学报,2015,23(05):46-52.
1.2 重组蛋白的构建和合成
根据spG的C片段以及链霉亲和素基因序列,利用Primer5软件设计引物,分别在上游和下游引物中加入酶切位点,引物序列如表1所示。
表1 扩增引物
转入BL21中。
1.3 重组质粒鉴定
对重构的重组蛋白序列进行进行PCR反应,反应体系如下:
将各组分混匀后,短暂离心置于PCR仪中进行反应,反应参数如下:
退火温度如下表:
对PCR产物进行电泳鉴定(图2)和双酶切鉴定(图3),测序鉴定结果显示与设计的序列一致。目的基因全片段大小约为680bp。C3片段大小约为170bp,SA片段大小约为480bp。
(C3片段PCR对应上下游引物:SEQ ID NO.1、2;SA PCR对应上下游引物:SEQ IDNO.3、4;全片段PCR对应上下游引物:SEQ ID NO.1、4)。
实施例2重组蛋白的表达和纯化
2.1 重组质粒的表达
时相:
(1)挑取适量含有目的片段的BL21穿刺菌,接种于5ml含Kan+的LB液体培养基中,置于37℃震荡培养箱中,250rpm震荡培养。
(2)当生长至对数期(OD600约为0.6时),加入终浓度为0.1mmol/L的IPTG进行诱导表达。在诱导表达前、诱导表达后1h、2h、4h、6h、8h分别取0.5ml菌液,应用SDS-PAGE电泳分析最佳诱导时间。
SDS-PAGE分析显示(图4),重组质粒pET-28a(+)-C3-SA在大肠杆菌BL21(DE3)中成功表达,且1mmol/L IPTG诱导后1-8h表达量随着时间的增长变化不明显,诱导2h后表达量达到最高并趋于稳定。当诱导时间超过4h时,杂蛋白的表达量不断增加,使得目的蛋白
的表达量减少,所以选择诱导时间为2h时最佳。
大量表达:
(1)挑取适量含有目的片段的BL21穿刺菌,接种于150ml含Kan+的LB液体培养基中,置于37℃震荡培养箱中,250rpm震荡培养。
(2)当生长至对数期(OD600约为0.6时),加入终浓度为0.1mmol/L的IPTG进行诱导表达,将诱导后的菌液 5000rpm离心15min弃上清,沉淀用10ml Binding buffer重悬,反复冻融三次后,冰浴超声破碎25min(超2s隔9s)后5000rpm离心15min,收集沉淀和上清。
(3)将离心后的沉淀用5ml 8mol 尿素重悬,冰上溶解2h,重复上述离心步骤,收集上清。
(4)将超声后上清、沉淀重悬后上清,分别加入等体积蛋白电泳缓冲液,用SDS-PAGE电泳分析表达产物的可溶性。
2.2重组蛋白的纯化
使用Ni-NTA Hisbind Resin对重组蛋白进行纯化(Ni-NTA Hisbind Resin序列号:70666-3),按照试剂盒说明书进行操作,简易操作步骤如下:
(1)取5ml树脂加入新空柱中静置平衡,当液面降到树脂表面时,依次进行以下步骤;
(2)用2CV ddH2O*2,charge buffer *3,binding buffer*2;
(3) 上样前留50ul;
(4) 3CV Binding Buffer*3(刚加上需要留50ul下清);
(5) 3CV Wash Buffer*2,同留50ul;
(6) 2CV Elution Buffer*2,同留50ul;
(7) 少量Strip Buffer(3ml),反复上样,全留,同取50ul。
(8) 将上述收集的溶液进行SDS-PAGE电泳,分析蛋白纯化情况。
SDS-PAGE分析显示(图5),重组质粒pET-28a(+)-G-SA表达的蛋白在超声上清和沉淀中均存在,沉淀中的蛋白含量高于上清中的蛋白含量,表明该蛋白具有一定的水溶性。重组蛋白超声上清经过Ni-NTAHisbindResin纯化后,在经过Strip Buffer洗脱后,得到纯化。
实施例3重组蛋白活性鉴定
3.1 Western blotting检测重组蛋白与IgG的结合活性
(1)将纯化后的蛋白进行SDS-PAGE电泳,之后将蛋白转移至NC膜上,130mA,75min。
(2)将NC膜浸泡于PBST稀释的5%脱脂奶粉中,室温封闭2h。
(3)将封闭后的NC膜用PBST洗涤三次,每次5min。
(4)将NC膜用HRP标记的山羊抗小鼠IgG,兔抗山羊IgG,小鼠抗兔IgG,驴抗山羊IgG,生物素(用PBST 1:2000稀释)作为抗体,室温孵育1h。
(5)将孵育后的NC膜用PBST洗涤三次,每次10min。
(6)将NC膜用DAB双组份显色液试剂盒进行显色,显色后用流水冲洗,终止反应。
结果显示重组蛋白具有与兔IgG,驴IgG,鼠IgG,羊IgG,生物素等的结合能力。(图6)
3.3 ELISA法测定重组蛋白与不同物种IgG亲和常数
(1)用BCA法测定重组蛋白的浓度,并用商品化的标准蛋白G(SPG)作为对照。
(2)用包被液将蛋白从10μg/ml开始进行倍比稀释,共进行8个稀释度,以100μl每孔包被于96孔板上,每个浓度设置3个重复,4℃包被过夜。
(3)将96孔板用PBST洗涤三次,200μl每孔,每次5min。
(4)加入用PBST稀释的5%脱脂奶粉的溶液,150μl每孔,37℃封闭2h。
(5)将96孔板用PBST洗涤三次,200μl每孔,每次5min。
(6)将HRP标记的山羊抗小鼠IgG,兔抗山羊IgG,小鼠抗兔IgG,驴抗山羊IgG,生物素,用PBST按1:500、1:1000、1:2000、1:4000进行倍比稀释,100μl每孔,37℃孵育2h。
(7)将96孔板用PBST洗涤三次,200μl每孔,每次5min。
(8)加入TMB进行显色,100μl每孔,室温反应15min。
(9)加入2mol/L H2SO4终止反应,30μl每孔,读取OD450值。
以OD450值作为纵坐标,以抗原浓度的对数值作为横坐标,进行曲线拟合,亲和曲线如图7所示根据拟合的曲线,代入相应的公式中,分别计算亲和常数Ka,将得到的Ka值取其平均数,获得了重组蛋白的亲和常数值,结果见表2。
表2 G-SA与不同物种抗体、生物素的亲和常数
抗体 | 亲和常数 |
山羊 | 4.12*10^4 |
驴 | 1.26*10^5 |
兔 | 5.12*10^4 |
鼠 | 1.03*10^5 |
HRP-bio | 7.73*10^4 |
结果显示该重组蛋白同时具有SPG与不同物种IgG结合的能力以及SA的与生物素结合的功能,是一种新型的双功能重组蛋白。经western和ELISA测亲和常数均显示重组蛋白具有与IgG的结合能力和与生物素的结合能力。
实施例4 日本血吸虫病新型ELISA诊断试剂盒的优化
制备日本血吸虫虫卵可溶性抗原。用碳酸盐缓冲液稀释虫卵可溶性抗原稀释至终浓度10ug/ml。每孔100ul包被于酶标板中,4℃包被过夜,PBST洗涤3-次,每次5min;以PBST配制5%脱脂奶粉,每孔200ul,37℃2h,PBST洗涤3-5次,每次5min;牛日本血吸虫阴阳性血清1:100稀释,每孔80ul,37℃2h,PBST洗涤3-5次,每次5min;将重组蛋白以5%脱脂奶粉稀释至0.125ug/ml,0.25ug/ml,0.5ug/ml,1ug/ml,2ug/ml,4ug/ml,8ug/ml,16ug/ml,加入bio-HRP,AV-HRP至稀释度为1:1000,1:2000,37℃孵育2h。得rSPG-SA-biotin-HRP-AV-HRP混合液;将混合液每孔60ul加入酶标板中,37℃孵育2h,PBST洗涤3-5次,每次5min。TMB显色液每孔100ul,显色15min,2M H2SO4每孔50ul终止反应,读取OD450nm值。模式图如图8所示,结果见图9,结果如下,最适重组蛋白浓度确定为16ug/ml,此时P/N值最大,为9.649。
优化后BSA系统检测血吸虫病效果分析
实验确定的最佳条件如下,抗原包被浓度为10ug/ml;封闭液为5%脱脂奶粉;重组蛋白用5%脱脂奶粉稀释至16ug/ml,加入bio-HRP,AV-HRP至稀释度为1:1000,1:2000,37℃孵育2h,得16ug/ml rSPG-SA-biotin-HRP-AV-HRP混合液。以上述实验确定的最佳条件,同时检测兔,牛,羊日本血吸虫阴阳性血清,血清进行梯度稀释,检测此实验方法同时进行多物种检测的可行性及其灵敏度,结果见图10,兔日本血吸虫阳性血清最高可稀释至1:800仍检出阳性,而牛、羊日本血吸虫阳性血清在1:3200稀释度是仍检出阳性,说明此法灵敏度极高,具有较高的实用价值。
以上述实验确定最佳条件,诊断抗原为日本血吸虫虫卵可溶性抗原(SEA),其余操作同前,检测牛日本血吸虫阳性血清,牛大片吸虫阳性血清,羊东毕吸虫阳性血清,羊捻转血矛阳性血清,设牛,羊标准阴性血清对照,检测此实验方法与捻转血矛线虫,东毕吸虫,大片吸虫的交叉性,结果见图11,在1:50血清稀释度时,牛大片吸虫阳性血清检出为阳性,P/N值≥2.1,但是随着血清稀释度的提高,牛大片吸虫、羊捻转血矛线虫,羊东毕吸虫阳性血清均检测为阴性,说明血清稀释度在1:100及以上时,日本血吸虫为诊断抗原的新型ELISA检测法与捻转血矛线虫、东毕吸虫。大片吸虫没有交叉性;牛日本血吸虫阳性血清检测稀释至1:1600仍检出阳性,提示,此法特异性较好。
<110>中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心)
<120>一种兼有IgG结合活性及生物素结合活性的双功能蛋白及其ELISA试剂盒
<160> 8
<170> PatentIn version 3.5
<210> 1
<211> 19
<212> DNA
<213> C区上游引物
<400> CCG GGA TCC ACT TAC AAA C
<210>2
<211> 19
<212> DNA
<213> C区下游引物
<400> GCT ACCACC ACC ACC ACT G
<210>3
<211> 25
<212> DNA
<213> SA区上游引物
<400> GAT CCG AGC AAA GAT AGC AAA GCC C
<210>4
<211> 23
<212> DNA
<213> SA区下游引物
<400> GGC CTC GAG TTA TTG CTG AAC AG
<210>5
<211>675
<212> DNA
<213> 重组蛋白G-SA的核苷酸序列
<400>ACTTACAAACTTGTTATTAATGGTAAAACGCTGAAGGGTGAAACCACCACCAAAGCGGTGGATGCCGAAACCGCGCAGAAGGCCTTTAAGCAGTACGCCAACGACAATGGCGTGGATGGTGTTTGGACCTACGACGACGCGACCAAAACCTTTCGTGTTACCGAAGGCGGTGGTGGCAGTGGTGGTGGTGGTAGCGATCCGAGCAAAGATAGCAAAGCCCAAGTGAGTGCCGCGGAAGCCGGCATTACGGGTACGTGGTACAACCAGCTGGGCAGCACCTTCATTGTTACGGCGGGTGCCGATGGTGCCCTCACCGGTACGTACGAAAGCGCGGTTGGCAATGCCGAAAGCCGTTACGTGCTGACCGGTCGTTATGATAGTGCGCCAGCGACCGATGGTAGTGGTACCGCGCTGGGTTGGACCGTTGCGTGGAAGAACAACTACCGCAATGCCCATAGCGCCACGACGTGGAGCGGTCAGTACGTTGGCGGTGCCGAAGCCCGTATCAATACGCAGTGGCTGCTGACCAGCGGTACGACCGAAGCGAATGCGTGGAAAAGTACGCTGGTGGGCCACGATACGTTCACCAAGGTGAAGCCAAGCGCCGCGAGCATCGATGCGGCCAAAAAAGCCGGCGTGAATAATGGCAACCCTCTAGACGCTGTTCAGCAATAA
<210>6
<211>165
<212>DNA
<213> 重组蛋白SPG片段的核苷酸序列
<400>ACTTACAAACTTGTTATTAATGGTAAAACGCTGAAGGGTGAAACCACCACCAAAGCGGTGGATGCCGAAACCGCGCAGAAGGCCTTTAAGCAGTACGCCAACGACAATGGCGTGGATGGTGTTTGGACCTACGACGACGCGACCAAAACCTTTCGTGTTACCGAA
<210>7
<211>480
<212> DNA
<213> 重组蛋白SA片段的核苷酸序列
<400>GATCCGAGCAAAGATAGCAAAGCCCAAGTGAGTGCCGCGGAAGCCGGCATTACGGGTACGTGGTACAACCAGCTGGGCAGCACCTTCATTGTTACGGCGGGTGCCGATGGTGCCCTCACCGGTACGTACGAAAGCGCGGTTGGCAATGCCGAAAGCCGTTACGTGCTGACCGGTCGTTATGATAGTGCGCCAGCGACCGATGGTAGTGGTACCGCGCTGGGTTGGACCGTTGCGTGGAAGAACAACTACCGCAATGCCCATAGCGCCACGACGTGGAGCGGTCAGTACGTTGGCGGTGCCGAAGCCCGTATCAATACGCAGTGGCTGCTGACCAGCGGTACGACCGAAGCGAATGCGTGGAAAAGTACGCTGGTGGGCCACGATACGTTCACCAAGGTGAAGCCAAGCGCCGCGAGCATCGATGCGGCCAAAAAAGCCGGCGTGAATAATGGCAACCCTCTAGACGCTGTTCAGCAATAA
<210>8
<211>30
<212> DNA
<213>连接序列的核苷酸序列
<400>GGCGGTGGTGGCAGTGGTGGTGGTGGTAGC
Claims (4)
1.一种兼有IgG 结合活性及生物素结合活性的双功能蛋白G-SA,其特征在于所述双功能蛋白G-SA包括链球菌蛋白G片段和SA蛋白,所述双功能蛋白G-SA由SEQ ID NO.5所示的核苷酸序列编码。
2.一种如权利要求1所述的双功能蛋白G-SA的表达方法,所述方法为:(1)根据链球菌蛋白G蛋白的C3片段的基因序列,从含有C3片段的菌液中PCR扩增出C3序列;
(2)通过PCR从含有SA序列的菌液中扩增出SA序列;
(3)先将扩增出的SA序列双酶切后,连接到表达载体上,经鉴定后再将C3片段双酶切后连接上去,构建重组蛋白的原核表达质粒;
(4)将所述表达载体转入表达宿主中培养,活化至对数生长期后加入诱导剂表达蛋白;
经破碎、纯化后制得双功能蛋白G-SA。
3.如权利要求2所述的表达方法,其中,步骤(1)中使用的引物对如SEQ ID NO.1、2所示;
步骤(2)中使用的引物对如SEQ ID NO.3、4所示。
4.一种用于免疫分析的产品,所述产品包括权利要求1所述的双功能蛋白G-SA。
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