CN113248627B - 一种增强猪流行性腹泻免疫的抗原及其制备方法和应用 - Google Patents
一种增强猪流行性腹泻免疫的抗原及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及动物免疫学技术领域,尤其涉及一种增强猪流行性腹泻免疫的抗原及其制备方法和应用。该抗原为猪流行性腹泻病毒COE结构域与双载体Fc‑Ii融合蛋白,即Fc‑COE‑Ii融合蛋白。本发明构建串联小鼠IgG Fc和Ii的双载体(Fc‑Ii),利用FcRn介导IgG Fc跨越黏膜屏障进入体内和Ii促进抗原的递呈,从而更加有效的激发机体的局部粘膜免疫和全身性免疫反应。为开发新的猪流行性腹泻疫苗提供了理论依据并奠定了良好的基础,为新型粘膜疫苗的研制也提供了一种新的策略。
Description
技术领域
本发明涉及动物免疫学领域,尤其涉及一种增强猪流行性腹泻免疫的抗原及其制备方法和应用。
技术背景
猪流行性腹泻(Porcine epidemic diarrhea,PED)由猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)引起的猪的一种接触性肠道传染病,是导致世界各养猪国家仔猪早期死亡的重要疫病。该病特点是水样腹泻、脱水、呕吐,哺乳仔猪发病率可达到100%,大规模爆发后仔猪死亡率高达90%,在亚洲、欧洲和美国的养猪场均有发生。
PED作为一种高传染性肠道疫病,国内外主要针对PEDV灭活苗或弱毒苗进行了研究,但免疫保护效果不佳。因缺乏有效的疫苗,该病仍在世界范围内发生,给国内外养猪业造成了巨大的经济损失。
黏膜是许多病原菌进入机体的首要门户,要防止传染病从黏膜表面进入机体,必须诱导黏膜和全身的免疫反应,以应对早期感染和病原体的传播。黏膜上皮细胞和固有层内的免疫效应细胞之间的密切联系表明,通过黏膜表面输送免疫原是实现黏膜免疫和潜在的全身免疫的理想途径。
猪流行性腹泻病毒可以通过呼吸道黏膜表面感染机体,但目前仍缺乏可以抵御这种通过黏膜侵入机体的病原体的高效疫苗,如何通过疫苗引起猪机体的局部黏膜免疫和全身性免疫反应,降低猪流行性腹泻的发病率,是现有技术亟待解决的问题。
发明内容
为了解决现有技术中存在的问题,本发明的目的之一是提供一种用于增强猪流行性腹泻免疫的抗原,通过双载体Fc-Ii介导抗原结构域,可以增强接种猪产生特异性免疫应答,从而为研制出新型的PEDV疫苗奠定基础。
所述用于增强猪流行性腹泻免疫的抗原为猪流行性腹泻病毒COE结构域与双载体Fc-Ii融合蛋白,即Fc-COE-Ii融合蛋白,其氨基酸序列如SEQ ID NO.2 所示。
优选的,所述Fc-COE-Ii融合蛋白由pET-32a-Fc-COE-Ii重组质粒经感受态细胞表达得到,所述pET-32a-Fc-COE-Ii重组质粒的核苷酸序列如SEQ ID NO.3 所示。
本发明的另一目的在于提供一种制备上述用于增强猪流行性腹泻免疫的抗原的方法,包括如下步骤:
S1.分别提取鼠脾脏组织和猪肠道流行性腹泻病料组织样品的总RNA,并分别进行总RNA反转录得到cDNA,备用;
S2.通过PCR方法利用S1中cDNA扩增得到PEDV COE基因片段与小鼠 IgG Fc基因片段,测序确证;
S3.将PEDV COE基因片段与小鼠IgG Fc基因片段和pM-cherry-N1-Ii质粒串联,构建pET-32a-Fc-COE-Ii重组质粒;
S4.将pET-32a-Fc-COE-Ii重组质粒转化进感受态细胞中,所述感受态细胞为大肠杆菌BL21(DE3)菌种,利用大肠杆菌表达系统表达Fc-COE-Ii融合蛋白,该Fc-COE-Ii融合蛋白即为所需抗原。
优选的,所述S2中PCR为:以小鼠IgG2a重链基因序列设计一对Fc引物,引物序列如SEQ ID NO.4和SEQ ID NO.5所示,以猪流行性腹泻病毒S基因序列设计一对COE引物,COE引物序列如SEQ ID NO.6和SEQ ID NO.7所示,利用所述Fc引物和COE引物对S1得到的cDNA进行扩增。
优选的,对上述扩增产物先进行琼脂糖凝胶检测,再对含目的基因片段的胶回收,回收后的胶经DNA纯化柱离心、洗脱后,得到所述PEDV COE基因片段与小鼠IgG Fc基因片段。
优选的,所述S3的具体步骤为:
S31.利用含有Linker特异性引物Fc-up/Fc-down扩增得到Fc-Linker片段,利用含有Linker特异性引物COE-up/COE-down扩增得到Linker-COE片段;
S32.以Fc-Linker片段与Linker-COE片段为模板,Fc-up和COE-down为引物,经PCR扩增得到Fc-COE基因;
S33.将扩增的Fc-COE基因片段和pET-32a原核表达载体分别利用BamHI 和EcoRI进行双酶切处理,并将得到的Fc-COE基因片段和pET-32a载体片段连接得到重组质粒pET-32a-Fc-COE;
S34.以pET-32a-Fc-COE和pM-cherry-N1-Ii质粒为模版,以含有Linker 1 特异性引物Fc-COE-up/Fc-COE-down扩增得到Fc-COE-Linker片段,以含有 Linker 2特异性引物Ii-up/Ii-down扩增得到Linker-Ii片段;
S35.以Fc-COE-Linker片段与Linker-Ii片段为模板,以Fc-COE-up和 Ii-down为引物,经PCR扩增后,得到Fc-COE-Ii基因片段;
S36.利用BamHI和EcoRI双酶切处理Fc-COE-Ii和pET-32a表达载体,琼脂糖凝胶电泳和胶回收,获取Fc-COE-Ii基因片段和pET-32a载体片段并连接,得到pET-32a-Fc-COE-Ii重组质粒;
优选的,所述Linker 1和Linker 2均为富含甘氨酸和丝氨酸的Linker[(G4S)3],其中Linker 1的核苷酸序列如SEQ ID NO.8所示,Linker 2的核苷酸序列如SEQ ID NO.8所示,
优选的,所述连接均使用Ligation high Ver.2试剂16℃金属浴连接。
优选的,所述特异性引物Fc-up/Fc-down的序列如SEQ ID NO.9/SEQ ID NO.10所示;所述特异性引物COE-up/COE-down的序列如SEQ ID NO.11/SEQ ID NO.12所示;所述特异性引物Fc-COE-up/Fc-COE-down的序列如SEQ ID NO.13/SEQ ID NO.14所示;所述特异性引物Ii-up/Ii-down的序列如SEQ ID NO.15/SEQ ID NO.16所示。
优选的,所述S4制备融合蛋白后,还包括融合蛋白提取与纯化步骤,所述融合蛋白提取为将诱导表达该融合蛋白的大肠杆菌菌液离心并收集沉淀;所述纯化为镍柱亲和层析纯化。
本发明的再一目的是提供一种使用上述用于增强猪流行性腹泻免疫的抗原的方法,该方法为:将Fc-COE-Ii融合蛋白与黏膜免疫佐剂以体积比1:1的比例混匀,以滴鼻免疫法接种所需个体,用量为20μg/只。
优选的,所述黏膜免疫剂为CPG ODN 1826。
本发明还提供一种用于使猪机体产生或增强猪流行性腹泻免疫力的制剂,所述制剂包含有效剂量的Fc-COE-Ii融合蛋白。
本发明的有益效果在于:
1)PEDV S糖蛋白是诱导机体产生中和抗体和提供免疫保护作用的主要结构蛋白,而COE结构域是S糖蛋白中一个高度保守的抗原结构域,可以诱导机体产生中和抗体,对猪产生有效的保护力。FcRn是识别和转运IgG Fc的特异性受体,利用FcRn的受体功能,将IgGFc片段作为载体,与病原体保护性抗原构成IgG Fc融合蛋白,通过IgG传递的天然途径,突破上皮细胞屏障,增强抗原免疫应答作用。MHCII类分子恒定链(Ii)具有促进抗原递呈作用,可以作为免疫载体,与抗原肽连接后,能够增强免疫应答。本专利构建串联小鼠IgG Fc和Ii的双载体(Fc-Ii),利用FcRn介导IgG Fc跨越黏膜屏障进入体内和Ii促进抗原的递呈,从而更加有效的激发机体的局部粘膜免疫和全身性免疫反应。
2)本发明串联IgG Fc片段和Ii链,构建双载体融合抗原,通过实验证明, FcRn可介导与IgG Fc片段融合的PEDV COE穿过机体呼吸道粘膜屏障,并能有效的激发局部粘膜免疫和全身性免疫反应;MHC II类分子恒定链(Ii)具有促进抗原递呈的作用,Ii链与PEDVCOE抗原表位串联,可以有效增强机体免疫应答。因此,本发明为开发新的猪流行性腹泻疫苗提供了理论依据并奠定了良好的基础,为新型粘膜疫苗的研制也提供了一种新的策略。
附图说明
图1为Fc-Linker基因片段与Linker-COE基因片段的PCR扩增结果,图中 M为DNAMarker(DL2000);1为Linker-COE片段PCR扩增结果;2为Fc-Linker 片段PCR扩增结果;
图2为Fc-COE基因片段的PCR扩增结果,其中M为DNAMarker(DL2000); 1为Fc-COE基因的PCR扩增结果;
图3a为重组质粒pET-32a-Fc-COE菌液PCR鉴定结果,图3b为酶切鉴定结果,其中M为DNAMarker(DL2000);图3a中1-6为多个样品的 pET-32a-Fc-COE菌液PCR;图3b中1为pET-32a-Fc-COE重组质粒EcoRI和 SalI酶切;2为pET-32a空载体EcoRI和SalI酶切;
图4为Fc-COE-Linker与Linker-Ii基因的PCR扩增结果,其中M为DNA Marker(DL2000);1为Fc-COE-Linker基因的PCR扩增结果;2为Linker-Ii基因的PCR扩增结果;
图5为Fc-COE-Ii基因的PCR扩增结果,其中M为DNA Marker(DL2000); 1为Fc-COE-Ii基因的PCR扩增结果;
图6a为重组质粒pET-32a-Fc-COE-Ii菌液PCR鉴定结果,图6b为酶切鉴定结果,其中图a的1-5为pET-32a-Fc-COE-Ii菌液PCR;图b中1为 pET-32a-Fc-COE-Ii重组质粒EcoRI和SalI酶切,2为pET-32a空载体EcoRI和SalI酶切;
图7为融合蛋白的大量表达及可溶性鉴定结果,其中M为Protein Marke;泳道1-3分别代表了pET-32a-Fc-COE-Ii未诱导、诱导超声后的上清和沉淀结果;
图8为融合蛋白经His树脂纯化后SDS-PAGE检测结果,其中M为Protein Marker;泳道1-4分别为Fc-COE-Ii融合蛋白His树脂纯化的四个样品结果;
图9为通过间接ELISA方法测定其抗PEDV的IgG抗体水平动态变化结果,图9A显示了血清中IgG抗体水平;图9B显示了鼻腔冲洗液IgG抗体水平;图9C显示了肺脏冲洗液IgG抗体水平;图9D显示了肠腔冲洗液IgG抗体水平;图中数据以平均值±标准差表示(n=3),不同的小写字母表示同一检测时间各组间的差异显著(P﹤0.05);
图10为小鼠呼吸道、消化道、血清sIgA的水平测定结果,图10A为小鼠血清、图10B为小鼠鼻腔冲洗液、图10C为小鼠肺脏冲洗液、图10D为小鼠肠腔冲洗液中sIgA抗体水平检测结果。实验数据以平均值±标准差表示(n=3),不同的小写字母表示同一检测时间各组间的差异显著(P﹤0.05)。
图11为鼠血清中的IFN-γ、IL-2、IL-4和IL-10含量测定,图11A、图11B、图11C、图11D分别为鼠血清中IFN-γ、IL-2、IL-4和IL-10含量检测结果;图中数据以平均值±标准差表示(n=3),不同的小写字母表示同一检测时间各组间的差异显著(P﹤0.05)。
具体实施方式
为了便于理解,下面结合实施例对本发明的技术方案做出更为具体的说明:
实施例1:试验材料
1.病料及实验动物来源
猪流行性腹泻肠道组织病料由安徽农业大学提供;清洁级6-8周龄雌性Balb/ c小鼠购于安徽医科大学实验动物中心。
2.菌株与质粒
原核表达载体pET-32a、PGEX-6P-1以及大肠杆菌工程菌E.coli Rosetta(DE3)、E.coli BL21(DE3)、DH5α等菌株由实验室保存。小鼠pcmv-myc-N1-Ii质粒由安徽农业大学提供。
3.培养基与主要抗生素的配置
配制LB液体培养基:分别称取酵母提取粉5g,胰蛋白胨粉末10.0g,NaCl 晶体10.0g,溶于800μL dd H2O,定容至1L,使用1mol/L NaOH溶液调pH至 7.4,121℃高压蒸汽灭菌20min,4℃冰箱保存备用。
配制LB固体培养基:称取40g的LB琼脂干粉,溶于800μL dd H2O,加热溶解后,定容至1L,使用1mol/L NaOH溶液调pH至7.4,121℃高压蒸汽灭菌 20min,4℃冰箱保存备用。
氨苄青霉素溶液的配置:称取2g青霉素干粉,溶于15mL无菌超纯水,定容到20mL,用孔径0.22μm滤嘴过滤分装,-20℃冰箱保存备用。
卡那霉素溶液的配置:称取2g卡那霉素干粉,溶于15mL无菌超纯水,定容到20mL,用孔径0.22μm滤嘴过滤分装,-20℃冰箱保存备用。
4.琼脂凝胶电泳相关试剂的配制
琼脂凝胶电泳相关试剂的配制:1×TAE的配制:量取10mL的50×TAE溶于480mL ddH2O中,定容到500mL,装瓶,常温保存。
1.5%琼脂糖凝胶的配制:称取4.5g琼脂糖干粉,溶于30mL 1×TAE溶液中,加热溶解后加入0.9μL核酸染料,混匀后倒板。
5.蛋白诱导表达与纯化相关试剂的配制
IPTG溶液的配制:精确称取0.24g IPTG干粉,溶于8mL高压灭菌后的dd H2O中,定容至10mL,用孔径0.22μm滤嘴过滤分装,-20℃冰箱保存备用。
0.25mol/L KCL溶液的配制:称取9.4g KCL晶体,溶于480mL dd H2O中,定容到500mL,装瓶,常温保存。
PBS缓冲溶液的配制:分别称取NaCl 4.0g,KH2PO4 0.1g,Na2HPO4·12H2O 1.45g,KCL晶体0.1g,溶于480mL dd H2O中,定容到500mL,pH调至7.4, 121℃高压灭菌20min,4℃冰箱保存备用。
6.纯化相关试剂的配制
结合/洗涤缓冲液的配制:称取NaCl 0.88g,Na2HPO4 0.28g,取足量各组分,溶于蒸馏水,充分混匀并定容至100mL,调节pH至7.0。
洗脱液的配制:称取0.75g glycine,混匀定容至100mL,pH调节至3.0。
中和液的配制:称取Tris 12.1g,溶于80mL dd H2O中,混匀后用盐酸调节 pH到8.5,定容至100mL。
使用前将缓冲液通过0.45μm滤膜过滤。
7.主要仪器
实施例2:双载体融合蛋白的构建和纯化
1.克隆PEDV-COE基因和小鼠IgG-Fc基因
1.1引物设计
参照NCBI-GenBank数据库中小鼠IgG2a重链基因序列(登录号:V00798.1, MousemRNA for gamma-2a-immunoglobulin heavy-chain GenBank),设计一对特异性引物:Fc-F/Fc-R,引物上游选用BamHI作为酶切位点,引物下游选用EcoRI 作为酶切位点。参照NCBI-GenBank数据库中猪流行性腹泻病毒S基因序列(登录号:MH991860.1,Porcine epidemicdiarrhea virus strain Y11-FJ2018 S protein(S)gene,complete CDs,S:4158bp,COE:1504-1926bp,423bp,设计一对特异性引物:COE-F/COE-R,引物上游选用EcoRI作为酶切位点,引物下游选用SalI作为酶切位点。
1.2鼠脾脏组织与猪肠道组织RAN提取
S1.将超低温冻存的鼠脾脏组织、肠道病料组织样品分别迅速转移至液氮预冷的研钵中,用研杵研磨组织,期间不断加入液氮,直至研磨成粉末状(无明显的可见颗粒),将研磨成粉末状的样品加入到含有裂解Buffer RL的1.5mL灭菌EP管中,用移液器反复吹打直至裂解液中无明显沉淀。
S2.将裂解液12,000rpm,4℃离心5min,小心吸取上清液到新的1.5mL无 RNA酶的EP管中,加入与液体等体积的70%乙醇,使用移液枪将溶液混合均匀。
S3.立即将混合液全部转入含RNA吸附柱的2mL EP管中,12,000rmp,离心1min弃滤液,将RNA吸附柱放回到2mL EP管中。
S4.将500μL Buffer RWA加入至RNA吸附柱中,12,000rmp离心30s,弃滤液。
S5.将600μL Buffer RWB加入至RNA吸附柱中,12,000rmp离心30s,弃滤液。
S6.DNase I消化:①DNase I反应液的配制:取5μL 10×DNase I Buffer,4μLRecombinant DNase I(RNase free,5U/μL),41μL RNase free d H2O到新的1.5mL 无RNA酶EP管中,混合均匀;②向RNA吸附柱膜中央加入50μL DNase I反应液,室温静置15min;③向RNA吸附柱膜中央加入350μL Buffer RWB,12,000 rpm离心30s,弃滤液。
S7.重复第e步操作。
S8.将RNA吸附柱重新安置于2mL EP管上,12,000rmp离心2min。
S9.将RNA吸附柱安置于1.5mL的无RNA酶EP管中上,在RNA吸附柱膜中央加入50~200μL RNase Free dd H2O,室温静置5min。
S10.12,000rmp离心2min,洗脱RNA,将第一次的洗脱液重新加回至RNA 吸附柱中,室温静置5min,12,000rmp离心2min洗脱RNA,得到高浓度的RNA。
1.3RNA反转录
S1.移液枪吸取3μL组织总RNA,于无RNA酶的PCR管中,按下表加入各组分:
S2.浴锅中65℃保温5min,冰上迅速冷却,将上表中反应液与下中组分混合,缓慢混匀。
S3.95℃保温5min,使酶失活,冰上放置,存放于-80℃超低温冰箱,备用。
1.4IgG-Fc和PEDV-COE基因编码区的扩增
用上述鼠脾脏组织的cDNA为模板,引物Fc-F和Fc-R进行鼠IgG-Fc的扩增;用上述猪小肠病料组织的cDNA为模板,引物COE-F和COE-R进行猪流行性腹泻病毒(PEDV)结构中S蛋白区域COE抗原表位(aa:499-638)的扩增,高保真酶PCR反应体系:
高保真酶反应条件:
1.5琼脂糖凝胶检测
根据基因片段的大小,配制琼脂糖凝胶当琼脂糖完全融化后,加入适量的核酸染料,混匀后倒入凝胶板,插上对应大小的梳子,待胶凝固后方可使用, PCR反应结束后进行核酸电泳检测。
1.6目的片段胶回收
(a)电泳结束后,将目的片段进行胶回收,切取含有目的DNA片段的琼脂糖凝胶条带,将切下的琼脂糖凝胶称重,按照每100mg琼脂糖凝胶加入100μL BD 溶液比列。水浴锅调至60℃,水浴10min,期间振荡混合3次,直至凝胶完全融化,将所获溶液置于DNA纯化柱中,静置2min,12,000rmp离心1min。
(b)加入500μL溶液PE,12,000rmp离心1min,弃滤液。
(c)重复上一步。
(d)12,000rmp离心3min,彻底洗去纯化柱中的液体。
(e)将离心柱置于新的1.5mL的离心管中,向纯化柱的中央处,悬空滴加40μLEluent溶液(60℃预热),静置2min,12,000rmp离心1min.;将收集的洗脱液再一次加回到纯化柱滤膜中央,静置2min,12,000rmp离心1min,将洗脱液收集并贮存-20℃冰箱。
2.重组质粒的构建
2.1感受态细胞的制备
试验所用到的表达目的蛋白的感受态主要是E.coli BL21(DE3)和E.coliRosetta(DE3)两种,感受态细胞制备方法如下:
将感受态菌株接种于固体培养基,四区划线分离,37℃培养12h。
挑取单菌落接入2mL LB液体培养基中,200rmp/min,37℃震荡培养12h。
将上述菌液按1:100接入10mL的LB培养基,200rmp/min,37℃震荡培养 3h,取培养好的10mL细菌在冰中静置20min后,将菌液分装到2mL的EP管中,4℃,4500rmp/min,离心3min,弃上清,加入600μL的0.1mol/L的CaCl2溶液重悬细菌,冰浴30min。
将冰浴好的菌液于4℃,4500rmp/min,离心2min后弃去上清,每管用50μL 的0.1mol/L的CaCl2溶液重悬沉淀,即为感受态细胞,加入50μL的30%甘油混匀后于-80℃冰箱冻存备用。
2.2Fc-Linker片段与Linker-COE片段PCR扩增
S1.利用含有Linker特异性引物Fc-up/Fc-down扩增得到Fc-Linker片段,利用含有Linker特异性引物COE-up/COE-down扩增得到Linker-COE片段;经PCR 扩增后,用1.5%的琼脂糖凝胶电泳检测PCR扩增产物,结果如图1所示,显示分别有一条450bp和740bp的条带,大小与预期相符。
S2.以Fc-Linker片段与PEDV COE基因片段为模板,Fc-up和COE-down为引物,经PCR扩增得到Fc-COE基因;经PCR扩增,1.5%的琼脂糖凝胶电泳检测扩增产物,结果如图2所示,显示有一条1100bp大小的条带,与预计Fc-COE 基因大小相符。
S3.将扩增的Fc-COE基因片段和pET-32a原核表达载体分别利用BamHI和 EcoRI双酶切处理,胶回收获得Fc-COE基因片段(1170bp)和pET-32a载体片段(5900bp)并连接得到重组质粒pET-32a-Fc-COE。
具体连接体系如下表所示:
将连接物混匀后放入金属浴中,16℃连接30min。
上述S3和S4中,对连接得到的重组质粒进行感受态细胞转化获得单个菌落,再进行菌液PCR鉴定、质粒提取并双酶切鉴定。结果如图3所示,菌液PCR 和双酶切后均出现与预期片段大小相符的条带,表明得到的重组质粒 pET-32a-Fc-COE符合要求。
S4.以pET-32a-Fc-COE和pM-cherry-N1-Ii质粒为模版,以含有Linker特异性引物Fc-COE-up/Fc-COE-down扩增得到Fc-COE-Linker片段,以含有Linker 特异性引物Ii-up/Ii-down扩增得到Linker-Ii片段;经PCR扩增后,用1.5%的琼脂糖凝胶电泳检测扩增产物,结果如图4所示,显示分别有一条1000bp和 750bp的条带,与预期的Fc-COE-Linker基因和Linker-Ii基因大小相等。
S5.以回收的Fc-COE-Linker片段与Linker-Ii片段为模板,以Fc-COE-up和 Ii-down为引物,经PCR扩增后得到Fc-COE-Ii基因片段,用1.0%的琼脂糖凝胶电泳检测扩增产物,如图5所示,结果显示有一条约1800bp的条带,与预计 Fc-COE-Ii基因大小相符。
S6.利用BamHI和EcoRI双酶切处理Fc-COE-Ii和pET-32a表达载体,琼脂糖凝胶电泳、胶回收,获取Fc-COE-Ii基因片段(1854bp)和pET-32a载体片段 (5900bp)并连接得到pET-32a-Fc-COE-Ii重组质粒。
具体连接体系如下表所示:
连接物混匀后放入金属浴中,16℃连接30min,得到重组质粒。
将得到的重组质粒pET-32a-Fc-COE-Ii菌液转化获得单个菌落,再进行菌液 PCR鉴定、质粒提取并双酶切鉴定。结果如图6所示,可以看出菌液PCR和双酶切后均出现与预期片段大小相符的条带,表明重组质粒pET-32a-Fc-COE-Ii成功构建。
3.转化及融合蛋白表达
3.1感受态细胞转化
S1.从-80℃超低温冰箱中取出E.coli Rosetta(DE3)感受态细胞,迅速插入冰中,待菌块融化后将15μL连接产物加到感受态细胞中,轻轻拨弹混匀,静置冰浴30min。
S2.42℃水浴90s后,迅速放回冰上并静置2min,向EP管中加入700μL不含抗生素的无菌LB液体培养基,混匀后将EP管放入振荡培养箱在37℃,200rmp,震荡培养1h,5,000rmp离心1min,弃上清留下大约100μL LB培养液吹打沉淀,将菌体重悬,将液体涂布于含Amp/LB固体培养基,将平板倒置放于于37℃培养箱过夜培养。
S3.菌液鉴定。从培养皿上挑取大小合适,形态圆润的单菌落转入含有 Amp/LB液体培养基中,200rmp,37℃振荡培养6小时,进行菌液PCR鉴定。 PCR鉴定为阳性的重组菌即为成功转化。
3.2融合蛋白的表达与检验
挑取已测序正确的重组菌的单菌落接种于含有100mg/L浓度Amp/LB液体培养基中,37℃过夜振荡培养。次日按照1:100稀释度接种新的Amp/LB液体培养基中,37℃振荡培养4h后,向培养物中加入IPTG溶液使其终浓度为 0.8mmol/L,37℃振荡培养5h,诱导融合蛋白表达。
将重组菌pET-32a-Fc-COE-Ii进行大量融合蛋白诱导,诱导后离心收集沉淀进行超声处理,超声结束后离心分出上清和沉淀,经SDS-PAGE凝胶电泳检测。如图7所示,融合蛋白主要存在于超声后的沉淀中,即目的蛋白主要以包涵体的形式表达,上清中则几乎没有目的蛋白说明表达的融合蛋白均不可溶。 SDS-PAGE结果说明pET-32a-Fc-COE-Ii融合蛋白的大量表达良好。
4.融合蛋白大量提取
4.1提取
将诱导表达后的菌液分装入50mL高压灭菌后的离心管,离心收集菌体沉淀,加入10mL PBS缓冲液重悬菌体沉淀,洗涤一次,收集菌体沉淀,加入10mL PBS 缓冲液重悬菌体沉淀,于冰水浴中超声破碎,超声破碎结束后,4℃,6,000rmp/min 离心10min,收集沉淀。
4.2包涵体变性与复性
S1.配制包涵体蛋白洗涤缓冲液:
配制裂解液:称取Urea 240g,溶于1×PBS缓冲液中,将pH调至7.4,用用0.45μm的滤膜过滤,常温保存。
配制复性缓冲液:称取一定量的Urea,溶于1×PBS缓冲液中使其Urea终浓度分别为6M、4M、2M、1M、0.5M、PBS,pH调至7.4,4℃保存。
配制His标签蛋白纯化缓冲液:
S2.处理透析袋:
把透析袋剪成适当长度(10-20cm)的小段,在大体积的2%(W/V)碳酸氢钠和1mmol/L EDTA(pH 8.0)中将透析袋煮沸10min,用蒸馏水彻底清洗透析袋,使透析袋始终浸没在含20%乙醇溶液内,4℃存在备用。
S3.镍柱再生:
用5倍柱床体积2M NaCl溶液淋洗柱子,再反向淋洗,最后用去离子水彻底清洗;20mM磷酸缓冲液,0.5M NaCl,50mM EDTA,调pH值为7.4,冲洗柱子,洗去残留的镍离子;蒸馏水反复清洗柱子,洗尽残留的EDTA。用NiSO4 (0.1M)2.63g,定容至100mL,过重力柱,再用蒸馏水彻底清洗没有结合的镍离子;20%的乙醇过重力柱,使填料保存在20%的乙醇环境中。
S4.His标签包涵体蛋白纯化
样品处理过程:将收集的菌体用平衡缓冲液悬浮,混匀后冰上放置1h,使包涵体溶解,7000rpm、4℃离心15min,收集上清,上柱前样品用0.45μm的滤膜过滤。
清洗填料:取适量的金属镍离子亲和层析介质装入重力柱,去离子水洗掉 20%乙醇,用初始缓冲液多次清洗后再用5-10个柱体积的结合缓冲液平衡重力柱,直到流出电导、pH不变时,加入适当体积的结合缓冲液制备50%浆液。
上样:将4mL样品加入重力柱,将样品和浆液混匀,室温下摇床上低速孵育1h,用2×1mL结合缓冲液洗涤并收集两种组分,用4×0.5mL洗脱缓冲液洗脱,将洗脱的组分收集在四个单独的EP管中,通过SDS-PAGE确认各组分的纯度。
检测结果如图8所示,可以看出经His标签蛋白纯化后,融合蛋白条带单一,表明获得的蛋白纯度较高。
实施例3:双载体融合蛋白滴鼻免疫
1.实验材料与试剂
Balb/c小鼠,6-8周龄,雌性,购自安徽医科大学实验动物中心。
CPG ODN1826(5'-TCCATGACGTTCCTGACGTT-3')由生工生物工程(上海) 股份有限公司合成,全链硫代磷酸化修饰,PAGE纯化。
主要试剂和仪器:
2.实验方法
清洁级雌性Balb/c小鼠,6~8周龄,将Balb/c小鼠随机分为5组,每组8 只。将纯化后的Fc-COE-Ii融合蛋白以及通过相同组织材料制备的非双载体抗原 COE、Fc-COE、COE-Ii与黏膜免疫佐剂混匀,通过滴鼻免疫的方式免疫小鼠,免疫剂量为20μg/只;对照组设为PBS组,各组小鼠均在乙醚麻醉下滴鼻免疫,第0、14、21、28、35d各滴鼻免疫1次。下次免疫前,断食断水约6h后,每组随机抓取2只小鼠,摘眼球取血,分离血清并脱颈处死小鼠,用无菌PBS缓冲液冲洗鼻腔、肺脏和小肠黏膜,收集冲洗液,4℃,10000g离心10min,吸取上清液,-80℃保存备用。ELISA法测定血清IgG、sIgA,鼻腔冲洗液sIgA、小肠冲洗液sIgA以及血清中IFN-γ、IL-2、IL-4和IL-10水平。
3.实验材料处理及检测
免疫后14、21、28、35d各组提前禁食禁水6h后分别取2只小鼠,眼球采血断颈处死,无菌条件下采取外周血、鼻腔冲洗液、肺脏冲洗液、小肠冲洗液及脾脏,进行相关指标检测。
上述冲洗液的制备方法为:鼻腔冲洗液收集:各组小鼠眼球采血断颈处死后,将小鼠于75%的酒精中浸泡3-5min,全身消毒。仰卧,无菌眼科剪和眼科摄剪开颈部皮肤,暴露气管并分离气管,200μL PBS从气管断端向上冲洗鼻部,冲洗3次,共600μL,收集从鼻咽流出的冲洗液。冲洗液2000rmp/min离心10min,收集上清,-20℃保存待测。
肺脏冲洗液收集:开胸并暴露气管,从气管中部剪断,将肺和心脏完整取出,置一平皿内,用滤纸沾于肺表面,200μL PBS从气管断端冲洗肺,反复吹吸10次,重复3次共600μL,冲洗液2000rmp/min离心10min,收集上清,-20℃保存待测。
小肠冲洗液收集:开腹,游离小肠,取出喷门端至回盲部末端全小肠,置一平皿内,2mL PBS反复冲洗小肠3次,冲洗液2000r/min,离心10min,收集上清,-20℃保存待测。
4.相关免疫指标检测与分析
4.1间接ELISA方法检测血清及黏膜冲洗液液IgG抗体水平:
用COE、Fc-COE、COE-Ii和融合重组蛋白抗原Fc-COE-Ii与佐剂CPG ODN 1826滴鼻免疫Balb/c小鼠后,采集14、21、28、35d的外周血并分离血清,通过间接ELISA方法测定其抗PEDV的IgG抗体水平动态变化,检测方法如下:
S1.包被:用包被液将纯化后的融合蛋白包被到96孔酶标板上,经过摸索,抗原的最终稀释浓度定为5μg/mL,4℃包被过夜;S2.洗涤:弃孔中溶液,拍干,每次加入洗涤液100μL/孔,洗涤3次,每次3min,弃去洗涤液,在吸水纸上将酶栋板拍干;S3.封闭:每孔加PBS-5%脱脂奶粉(称取0.25g脱脂奶粉溶于5mL 稀释液)100μL/孔,37℃封闭1h,重复第二步洗涤操作;S4.加待测血清:将待检血清稀释200倍。每孔加入200μL稀释血清,同时设阴性对照和空白对照, 37℃孵育40min,重复第二步洗涤操作;S5.加酶标二抗:用稀释液1:5000稀释羊抗鼠IgG酶标抗体,100μL/孔,37℃孵育40min,重复第二步洗涤操作;S6. 加底物混合溶液显色:加入TMB显色液(碧云天),100μL/孔,避光,室温显色10min后取出;S7.终止反应:2mol/LH2SO4终止液100μL/孔;S8.测定。
检测结果如图9所示,可以看出,与对照组PBS相比,血清中各组峰值点平均抗体水平(OD450nm)分别是:0.42(COE组)、0.48(COE-Ii组)、0.56(Fc-COE 组)、0.65(Fc-COE-Ii组),免疫后21d-35d检测时间点,双载体抗原组平均抗体水平均高于与其余各组(除PBS组)(P﹤0.05);单载体(Fc、Ii)两组产生的特异性抗体差异不明显(P>0.05),免疫后14d-35d检测时间点,各组检测结果与对照组(PBS)相比,差异明显(P﹤0.05),表明双载体(Fc-Ii)抗原组可刺激机体产生更高水平的特异性抗体。
4.2ELISA方法检测血清、呼吸道及消化道特异性sIgA
将COE、COE-sIi、sFc-COE、sFc-COE-sIi融合蛋白抗原与黏膜佐剂CPG ODN 1826佐剂混合,滴鼻免疫Balb/c小鼠,采用ELISA方法检测各组融合蛋白抗原在免疫的小鼠黏膜冲洗液(鼻腔、肺脏、小肠)中和血清中sIgA抗体的水平,检测方法如下:
S1.将收集的鼻腔、肺脏及小肠冲洗液加入相应的微孔板中,100μL/孔,设置标准品孔和空白孔,用封板胶纸封住反应孔,室温孵育2h。S2.将板内液体弃掉,加400μL/孔洗涤液,重复洗涤三次,最后排干残留液体。S3.除空白孔外,在每孔加入100μL辣根过氧化物酶(HRP)标记的检测抗体,用封板胶纸封住反应孔,室温孵育1h,重复洗板三次。S4.每孔内加入底物A、B各50μL,室温避光孵育15min。S5.每孔内加入50μL终止液,酶标仪测量450nm的吸光值,以标准品的浓度作横坐标,对应的OD值作为纵坐标,绘制标准品的线性回归曲线,按曲线方程计算各样本的浓度值(pg/mL)。
检测结果如图10所示,可以看出,不论在血清、消化道还是呼吸道冲洗液中,随 着免疫的加强,机体产生的sIgA水平逐渐升高;在28d,各检测样本中 sIgA的含量达到最高,同时期,血清中检测到sIgA的含量高于呼吸道冲洗液和消化道冲洗液,血清中各组峰值点平均sIgA的含量分别是:COE:16.1pg/mL、 COE-sIi:18.8pg/mL、sFc-COE:20.2pg/mL、sFc-COE-sIi:21.7pg/mL。免疫后 21d-35d检测时间点,双载体(Fc-Ii)、单载体(Fc)的抗原组中,血清、消化道和呼吸道冲洗液中sIgA的含量高均高于单载体(Ii)和COE抗原组(P﹤0.05),单载体(Ii)抗原组与COE组抗体水平相当,无显著差异(P>0.05),各抗原组与PBS对照组相比,抗体水平差异显著(P﹤0.05)。
4.3血清中IFN-γ、白介素IL-2、IL-4和IL-10检测
用COE、Fc-COE、COE-Ii和融合重组蛋白抗原Fc-COE-Ii与佐剂CPG ODN 1826滴鼻免疫Balb/c小鼠后,采集14、21、28、35d的外周血并分离血清,检测血清中IFN-γ,步骤如下:
S1.将IFN-γ标准品(试剂盒内带)按说明书依次稀释,稀释度分别为 800pg/mL、400pg/mL、200pg/mL、100pg/mL、50pg/mL、0pg/mL,按顺序加入ELISA板中;S2.设空白对照孔、标准品孔和待测样品孔,加不同浓度标准品50μL 至ELISA板中,空白对照孔不加样品、IFN-γ抗体,待测样品孔中先加入40μL 样品,然后再加IFN-γ抗体10μL;S3.每孔加入酶标试剂50μL,空白对照孔除外,封板膜封板后置37℃温育30min;S4.小心揭掉封板膜,弃去液体甩干,每孔加满洗涤液,静置30s后弃去,如此反复5次,拍干,每孔先后分别加入显色剂A、B各50μL,混匀,37℃避光显色10min;S5.每孔加终止液50μL;S6.用空白孔调零,测定各孔OD450nm的值,用标准品的浓度为横坐标,以OD450nm的值作为纵坐标,绘制标准曲线,用标准品的浓度与OD值计算出标准曲线的直线回归方程式,将样品的OD值代入方程式将OD450nm值转换成IFN-γ的量 (pg/mL)。
按白介素IL-2、IL-4和IL-10试剂盒说明书操作,检测血清中白介素IL-2、 IL-4和IL-10,具体步骤参考上述IFN-γ检测步骤。
检测结果如图11所示,从图上可以看出,各抗原免疫14d后,血清中均产生了IFN-γ,并随着免疫的加强呈上升趋势,其中,各峰值点平均IFN-γ检测量分别为:COE:636.2pg/mL、COE-Ii:676.4pg/mL、Fc-COE:793.2pg/mL、 Fc-COE-Ii:853.6pg/mL,免疫后各检测时间点,Fc-COE-Ii组中IFN-γ的平均检测量显著高于其他组(P﹤0.05);同时,血清中IL-2的含量随着免疫的加强不断增加,免疫后各检测时间点,Fc-COE组、Fc-COE-Ii组中IL-4的平均检测量显著高于其他组(P﹤0.05);免疫中后期,Fc-COE-Ii组中,IL-10的平均检测量较其他高(P﹤0.05)。
实验证明,Fc-COE-Ii组抗原对机体的刺激强于Fc-COE组、COE-Ii组和 COE组,Fc-COE组、COE-Ii组强于COE组。因此,本发明制备的抗原效果明显,且双载体(Fc-Ii)抗原组对机体刺激效果强于单载体(Fc、Ii),单载体(Fc、 Ii)对机体的免疫强度优于“裸”COE抗原肽,具有更好的效果。
以上实施方式仅用以说明本发明的技术方案,而并非对本发明的限制;尽管参照前述实施方式对本发明进行了详细的说明,本领域的普通技术人员应当理解:凡在本发明创造的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明创造的保护范围之内。
序列表
<110> 安徽农业大学
<120> 一种增强猪流行性腹泻免疫的抗原及其制备方法和应用
<141> 2021-05-21
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Claims (6)
1.一种增强猪流行性腹泻免疫的抗原,其特征在于,该抗原为猪流行性腹泻病毒COE结构域与双载体Fc-Ii融合蛋白,即Fc-COE-Ii融合蛋白,其氨基酸序列如SEQ ID NO.1所示。
2.如权利要求1所述的一种增强猪流行性腹泻免疫的抗原,其特征在于,所述Fc-COE-Ii融合蛋白由pET-32a-Fc-COE-Ii重组质粒经感受态细胞表达得到,所述pET-32a-Fc-COE-Ii重组质粒的核苷酸序列如SEQ ID NO.2所示。
3.一种制备如权利要求1所述的抗原的方法,其特征在于,包括如下步骤:
S1.分别提取鼠脾脏组织和猪肠道流行性腹泻病料组织样品的总RNA,并分别进行总RNA反转录得到cDNA,备用;
S2. 通过PCR方法利用S1中cDNA扩增得到PEDV COE基因片段与小鼠IgG Fc基因片段,测序确认;
所述PCR为:以小鼠IgG2a重链基因序列设计一对Fc引物,引物序列如SEQ ID NO.3和SEQ ID NO.4所示,以猪流行性腹泻病毒S基因序列设计一对COE引物,COE引物序列如SEQID NO.5和SEQ ID NO.6所示,利用所述Fc引物和COE引物对S1得到的cDNA进行扩增;对扩增产物先进行琼脂糖凝胶检测,再对含目的基因片段的胶回收,回收后的胶经DNA纯化柱离心、洗脱后,得到所述PEDV COE基因片段与小鼠IgG Fc基因片段;
S3. 将PEDV COE基因片段、小鼠IgG Fc基因片段和pM-cherry-N1-Ii质粒串联,构建pET-32a-Fc-COE-Ii重组质粒;
具体步骤为:
S31. 利用含有Linker特异性引物Fc-up/Fc-down扩增得到Fc-Linker片段,利用含有Linker特异性引物COE-up/COE-down扩增得到Linker-COE片段;
S32. 以Fc-Linker片段与Linker-COE片段为模板,Fc-up和COE-down为引物,经PCR扩增得到Fc-COE基因;
S33. 将扩增的Fc-COE基因片段和pET-32a原核表达载体分别利用BamHI和EcoRI进行双酶切处理,并将得到的Fc-COE基因片段和pET-32a载体片段连接得到重组质粒pET-32a-Fc-COE;
S34. 以pET-32a-Fc-COE和pM-cherry-N1-Ii质粒为模版,以含有Linker 1特异性引物Fc-COE-up/Fc-COE-down扩增得到Fc-COE-Linker片段,以含有Linker 2特异性引物Ii-up/Ii-down扩增得到Linker-Ii片段;
S35. 以Fc-COE-Linker片段与Linker-Ii片段为模板,以Fc-COE-up和Ii-down为引物,经PCR扩增后,得到Fc-COE-Ii基因片段;
S36. 利用BamHI和EcoRI双酶切处理Fc-COE-Ii和pET-32a表达载体,琼脂糖凝胶电泳和胶回收,获取Fc-COE-Ii基因片段和pET-32a载体片段并连接,得到pET-32a-Fc-COE-Ii重组质粒;
所述Linker 1的核苷酸序列如SEQ ID NO.7所示,Linker 2的核苷酸序列如SEQ IDNO.8所示,所述连接均使用Ligation high Ver.2试剂16℃金属浴连接;
所述特异性引物Fc-up/Fc-down的序列如SEQ ID NO.9/ SEQ ID NO.10所示;所述特异性引物COE-up/COE-down的序列如SEQ ID NO.11/ SEQ ID NO.12所示;所述特异性引物Fc-COE-up/Fc-COE-down的序列如SEQ ID NO.13/ SEQ ID NO.14所示;所述特异性引物Ii-up/Ii-down的序列如SEQ ID NO.15/ SEQ ID NO.16所示;
S4. 将pET-32a-Fc-COE-Ii重组质粒转化进感受态细胞中,所述感受态细胞为大肠杆菌BL21(DE3)菌种,利用大肠杆菌表达系统表达Fc-COE-Ii融合蛋白,该Fc-COE-Ii融合蛋白即为所需抗原。
4.如权利要求3所述的方法,其特征在于,所述S4制备融合蛋白后,还包括融合蛋白提取与纯化步骤,所述融合蛋白提取为将诱导表达该融合蛋白的大肠杆菌菌液离心并收集沉淀;所述纯化为镍柱亲和层析纯化。
5.一种使用如权利要求1所述的抗原的方法,其特征在于,将Fc-COE-Ii融合蛋白与黏膜免疫佐剂以体积比1:1混匀,以滴鼻免疫法接种所需个体;所述黏膜免疫佐剂为CPG ODN1826。
6.一种提供或增强机体对猪流行性腹泻免疫力的制剂,其特征在于,所述制剂包含有效剂量的如权利要求1所述的Fc-COE-Ii融合蛋白,该Fc-COE-Ii融合蛋白的氨基酸序列如SEQ ID NO.1所示。
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Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2809354A1 (en) * | 2012-01-31 | 2014-12-10 | Curevac GmbH | Negatively charged nucleic acid comprising complexes for immunostimulation |
CN104262488A (zh) * | 2014-09-24 | 2015-01-07 | 普莱柯生物工程股份有限公司 | 一种融合蛋白及其疫苗组合物的制备与应用 |
CN104667296A (zh) * | 2015-02-04 | 2015-06-03 | 四川农业大学 | 一种预防猪流行性腹泻疾病的疫苗 |
WO2019022463A2 (ko) * | 2017-07-24 | 2019-01-31 | (주)제이비바이오텍 | 면역원성시스템 및 이를 포함한 동물 백신 |
CN109456412A (zh) * | 2018-04-10 | 2019-03-12 | 扬州优邦生物药品有限公司 | 一种猪流行性腹泻病毒基因工程疫苗及其制备方法 |
CN110093357A (zh) * | 2019-04-17 | 2019-08-06 | 仲恺农业工程学院 | 猪流行性腹泻病毒多表位抗原、编码基因、制备方法及应用 |
CN111454876A (zh) * | 2019-01-18 | 2020-07-28 | 安徽农业大学 | 一种猪流行性腹泻病毒感染猪小肠黏膜上皮细胞系的方法 |
-
2021
- 2021-05-21 CN CN202110556410.4A patent/CN113248627B/zh active Active
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2809354A1 (en) * | 2012-01-31 | 2014-12-10 | Curevac GmbH | Negatively charged nucleic acid comprising complexes for immunostimulation |
CN104262488A (zh) * | 2014-09-24 | 2015-01-07 | 普莱柯生物工程股份有限公司 | 一种融合蛋白及其疫苗组合物的制备与应用 |
CN104667296A (zh) * | 2015-02-04 | 2015-06-03 | 四川农业大学 | 一种预防猪流行性腹泻疾病的疫苗 |
WO2019022463A2 (ko) * | 2017-07-24 | 2019-01-31 | (주)제이비바이오텍 | 면역원성시스템 및 이를 포함한 동물 백신 |
CN109456412A (zh) * | 2018-04-10 | 2019-03-12 | 扬州优邦生物药品有限公司 | 一种猪流行性腹泻病毒基因工程疫苗及其制备方法 |
CN111454876A (zh) * | 2019-01-18 | 2020-07-28 | 安徽农业大学 | 一种猪流行性腹泻病毒感染猪小肠黏膜上皮细胞系的方法 |
CN110093357A (zh) * | 2019-04-17 | 2019-08-06 | 仲恺农业工程学院 | 猪流行性腹泻病毒多表位抗原、编码基因、制备方法及应用 |
Non-Patent Citations (3)
Title |
---|
lishment and application of a multiplex RT-PCR to differentiate wildtype and vaccine strains of porcine epidemic diarrhea virus;Dongxian He et al.;《Journal of Virological Methods》;20190706;第272卷;第1-6页 * |
Systemic and Oral Immunogenicity of Porcine Epidemic Diarrhea Virus Antigen Fused to Poly-Fc of Immunoglobulin G and Expressed in ΔXT/FT Nicotiana benthamiana Plants;Nguyen-Quang-Duc Tien et al.;《FRONTIERS IN PHARMACOLOGY》;20210430;第12卷;第1-13页 * |
基于S1蛋白核心中和表位区的猪流行性腹泻亚单位疫苗研究;王雅婷;《中国优秀博硕士学位论文全文数据库(硕士)农业科技辑》;20180215(第02期);第D050-492页 * |
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