CN116041497A - 一种猫杯状病毒(fcv)重组蛋白单克隆抗体及制备方法 - Google Patents
一种猫杯状病毒(fcv)重组蛋白单克隆抗体及制备方法 Download PDFInfo
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Abstract
本发明属于生物工程技术领域。本发明涉及一种重组蛋白,该重组蛋白包含猫杯状病毒(FCV)(Feline calicivirus,FCV)蛋白的两个优势抗原表位,为提高该重组蛋白在原核表达系统中的产量,采用大肠杆菌偏爱密码子将该重组蛋白氨基酸序列转换为对应的核苷酸序列,化学合成该核苷酸序列并构建重组表达载体。本发明还涉及用该重组蛋白免疫小鼠建立噬菌体库,经淘选筛选得到对应猫杯状病毒(FCV)蛋白单链抗体scfv序列,将得到的scfv序列构建成完整鼠IgG1抗体序列表达载体,通过瞬转HEK293F细胞表达单克隆抗体,纯化单克隆抗体并分别标记铕离子(Eu3+),通过正交实验确定最佳单抗配对组合,可用于猫上呼吸道感染的早期诊断。
Description
技术领域
本发明属于生物工程技术领域。具体来说,本发明涉及一种新的重组蛋白,涉及使用上述重组蛋白免疫小鼠建立噬菌体库,筛选得到特异性单链抗体scfv序列,还涉及将得到的scfv序列构建成真核表达载体表达猫杯状病毒(FCV)蛋白单克隆抗体,并应用于猫的上呼吸道感染的早期诊断。
背景技术
猫杯状病毒(Feline calicivirus,FCV)属于杯状病毒科的成员之一,是猫的一种重要病原。近年来,有高致病性的FCV毒株(VSD-FCV)在一些国家相继爆发,该毒株可引起感染猫全身性多系统病变,死亡率较高。猫杯状病毒(FCV)感染的主要症状是发热(40℃左右)、口腔溃疡(舌或者上下颚)、鼻腔炎症、结膜炎、间质性肺炎、多发性关节炎等。病猫精神欠佳、打喷嚏,口腔及鼻腔分泌物增多,流涎,眼鼻分泌物开始为浆液性、4~5天后为脓性,角膜发炎、可导致失明。口腔溃疡是为最显著的特征,以舌和硬腭、腭中裂周围明显,出现大面积的溃疡和肉芽增生,病猫进食困难,容易造成营养不良、脱水甚至死亡。猫杯状病毒(FCV)的潜伏期一般在二至三日,病毒毒力较强时,可发生肺炎,呼吸困难,肺部有干性或湿性啰音,三个月以下幼猫可因肺炎致死。杯状病毒感染如不继发其他病毒(传染性鼻气管炎病毒)、细菌性感染,7~10天后可自行恢复,但往往成为带毒猫。
疫苗接种是防治本病最有效方法,但现有疫苗的保护效果并不理想,临床上主要有对症治疗和支持治疗相结合的方法。在猫感染病毒初期,对于呼吸道感染情况,除使用抗病毒药物、干扰素和抗生素,防止继发感染以外,还需要进行吸氧、雾化治疗,帮助猫咪化痰止咳;当出现呼吸困难,可通过吸氧进行缓解,建议选用速诺和强力霉素这两种混合联合用药。因此,控制本病的根本措施,在于免疫预防和检疫。
目前获得猫杯状病毒(FCV)单克隆抗体主要是通过杂交瘤技术。但杂交瘤技术获得单抗具有一定的局限性:(1)针对动物毒性抗原、自身抗原、免疫耐受原和弱免疫原性抗原,由于无法产生有效免疫,故而不适合采用杂交瘤技术来制备抗体。(2)单克隆抗体固有的亲和性和局限的生物活性限制了它的应用范围。由于单克隆抗体不能进行沉淀和凝集反应,所以很多检测方法不能用单克隆抗体完成。(3)作为异源杂合二倍体细胞,候选杂交瘤克隆需要亚克隆后才能稳定。不及时亚克隆的候选杂交瘤细胞不稳定,容易丢失阳性克隆。而挑选阳性克隆,尤其是复杂的功能筛选,需要一定的时间窗口,窗口越长,功能筛选越成熟和可信。微量冻存候选克隆可以延长功能筛选时间窗口,但功能检测需要足够量的抗体蛋白,因此批量亚克隆仍然需要大的工作量。杂交瘤筛选失败后的应对方案是重新免疫或者重新融合(也受限于免疫后小鼠的饲养周期),必然产生时间成本。
因此制备猫杯状病毒(FCV)蛋白单克隆抗体用于猫杯状病毒(FCV)特异性识别检测成为早期诊断的主要方式。常规猫杯状病毒(FCV)蛋白单克隆抗体制备是将猫杯状病毒(FCV)蛋白单克隆细胞株制备Balb/c小鼠腹水,使用Protein A亲和层析法纯化单克隆抗体。但由于单只小鼠腹水产量不确定性且个体差异大,得到的抗猫杯状病毒(FCV)蛋白单克隆抗体批间差异大,使得检测准确性较差。
发明内容
为解决背景技术中制备单克隆抗体的不足之处,通过设计、合成猫杯状病毒(FCV)蛋白并通过建立噬菌体库和真核细胞表达来制备其单克隆抗体,以达到节省时间,而且能降低批间差异,提高检测准确性的目的。
为了实现上述目的。本申请提供了:
一种抗猫杯状病毒(FCV)特异性单链抗体scfv-3C5,包括轻链和重链,轻链可变区的氨基酸序列如SEQ ID NO.1所示;重链可变区的氨基酸序列如SEQ ID NO.2所示。
一种抗猫杯状病毒(FCV)特异性单链抗体scfv-5E2,包括轻链和重链,所述轻链可变区的氨基酸序列如SEQ ID NO.3所示;所述重链可变区的氨基酸序列如SEQ ID NO.4所示。
一种编码权利要求1所述的猫杯状病毒(FCV)特异性单链抗体scfv-3C5的基因,编码轻链可变区的核苷酸序列如SEQ ID NO.5所示;编码重链可变区的核苷酸序列如SEQ IDNO.6所示。
一种编码权利要求2所述的猫杯状病毒(FCV)特异性单链抗体scfv-5E2的基因,编码轻链可变区的核苷酸序列如SEQ ID NO.7所示;编码重链可变区的核苷酸序列如SEQ IDNO.8所示。
一种质粒载体,该质粒载体含有编码轻链可变区的核苷酸序列如SEQ IDNO.5所示。
一种质粒载体,该质粒载体含有编码重链可变区的核苷酸序列如SEQ IDNO.6所示。
一种质粒载体,该质粒载体含有编码轻链可变区的核苷酸序列如SEQ IDNO.7所示。
一种质粒载体,该质粒载体含有编码重链可变区的核苷酸序列如SEQ IDNO.8所示。
上述质粒载体在真核表达猫杯状病毒(FCV)单克隆抗体的应用,包括:
(a)通过PCR将上述轻链和重链核苷酸序列分别与鼠IgG1轻链恒定区和重链恒定区核苷酸序列桥接后酶切,并分别连接质粒载体,构建真核细胞表达载体;
(b)将步骤(a)中真核表达载体转染至HEK293F细胞表达得到猫杯状病毒(FCV)单克隆抗体;
(c)纯化单克隆抗体通过免疫荧光正交实验确定为最佳单抗配对组合。
有益效果:采用免疫抗体库筛选猫杯状病毒(FCV)蛋白单克隆抗体,同等免疫条件下,候选克隆越多,越容易筛选出高活性抗体,并且抗体库一旦建成,就可以无限期保存。原始噬菌体库就足以反复筛选,无需重新建库,筛选单抗时仅需取少量噬菌体颗粒。此外,亲和淘选的方式既可以特异性富集结合抗原的抗体,还可以尽量扣除与对照蛋白结合的抗体,有效提高筛选效率。(1)以猫杯状病毒(FCV)蛋白为靶抗原,分析并选择该抗原的两个特异性优势抗原表位,序列比对结果显示所选择的两个抗原表位与其他蛋白序列无明显同源性。(2)为了促进所选择优势抗原表位对Balb/c小鼠免疫系统的刺激,增强免疫效果,将所选择的两个优势抗原表位序列通过柔性片段串联形成重组蛋白氨基酸序列。(3)采用大肠杆菌偏爱密码子,将重组蛋白氨基酸序列转换为对应的核苷酸序列,以利于重组蛋白在大肠杆菌中的高效表达。(4)化学合成上一步骤得到的核苷酸序列,并通过酶切连接,将合成得到的核苷酸片段插入原核表达载体PET-32a,构建重组蛋白表达载体。(5)重组蛋白表达载体转化大肠杆菌ER2566感受态细胞,加氨苄青霉素抗性筛选培养基筛选得到重组蛋白表达菌株。(6)重组蛋白表达菌株大规模培养后,超声破菌并低温离心,取溶液上清通过镍琼脂糖亲和层析柱亲和层析,洗脱得到纯化重组蛋白。(7)纯化的重组蛋白多次免疫Balb/c小鼠后,取其脾脏分离淋巴细胞用来建立单链抗体scfv噬菌体展示库,使用抗猫杯状病毒(FCV)蛋白进行多轮淘选筛选最终得到能与重组猫杯状病毒(FCV)结合的单链抗体scfv序列。(8)将scfv序列构建成完整鼠IgG1表达载体并使用HEK293细胞表达单克隆抗体,使用Protein A亲和层析纯化单克隆抗体,并分别标记铕离子(Eu3+)。(9)正交实验筛选显示3C5单抗包被与5E2-Eu标记单抗配对检测猫杯状病毒(FCV)蛋白为最佳组合。
具体实施方案:以下实施例虽然对本发明的设计思路作了比较详细的文字描述,但是这些文字描述,只是对本发明设计思路的简单文字描述,而不是对本发明设计思路的限制,任何不超出本发明设计思路的组合、增加或修改,均落入到本发明的保护范围内。
实施例1:猫杯状病毒(FCV)蛋白优势抗原表位选择
以猫杯状病毒(FCV)蛋白为靶抗原,利用生物软件DNAssist2.0分析其抗原表位序列的亲水性及抗原性,选择A优势抗原表位和B优势抗原表位。同时,序列比较结果表明所选择的A、B两个优势抗原表位序列特异性高,与其他蛋白序列无明显同源性。
实施例2:猫杯状病毒(FCV)蛋白优势抗原表位串联
为增强所选择抗原表位对小鼠免疫系统的刺激以利于后续实验的进行,将猫杯状病毒(FCV)蛋白的A、B两个优势抗原表位序列分别重复再通过柔性片段(连续4个甘氨酸)连接,得到重组蛋白氨基酸序列。
实施例3:优化编码重组蛋白的核苷酸序列
为提高重组蛋白在大肠杆菌中的表达量,在重组蛋白氨基酸序列不变的前提下,根据大肠杆菌偏爱密码子将编码重组蛋白的氨基酸序列转化为对应的核苷酸序列,并在其上下游分别添加酶切位点BamHI和EcoRI对应的核苷酸序列,由杭州贤至生物科技有限公司合成。合成后的目的基因克隆于pMD19-T载体(宝生物工程大连有限公司)中。
实施例4:构建重组蛋白表达载体
用限制性内切酶BamHI和EcoRI(宝生物工程大连有限公司)于37℃分别双酶切含目的基因的pMD19-T载体和PET-32a载体(德国Novagen公司)12小时,酶切产物分别行1%琼脂糖凝胶电泳,并分别切胶回收目的基因和PET-32a载体(本发明所使用的胶回收试剂盒均购自宁波中鼎生物技术有限公司)。使用T4连接酶(宝生物工程大连有限公司)将回收的目的基因和PET-32a载体按一定比例于4℃连接12小时后,连接产物转化DH5α感受态细胞(杭州贤至生物科技有限公司),并涂布于含氨苄青霉素抗性(50μg/mL)的LB平板上,于37℃恒温培养12小时后,于平板上挑取单克隆菌株至含氨苄青霉素抗性(50μg/mL)的LB液体培养基,37℃恒温摇床培养12小时后,采用质粒纯化试剂盒(本发明所使用的质粒纯化试剂盒均购自于宁波中鼎生物技术有限公司)提取质粒,经BamHI和EcoRI双酶切鉴定后得到正确的重组表达载体。
实施例5:构建猫杯状病毒(FCV)蛋白表达菌株
将构建好的重组表达载体转化E.coli ER2566感受态细胞,并涂布于含氨苄青霉素抗性(50μg/mL)的LB平板上,于37℃过夜培养。次日挑取平板上单克隆菌株至含氨苄青霉素抗性(50μg/mL)的LB液体培养基,37℃恒温摇床培养8小时后,取1mL保存,剩余加诱导剂IPTG(异丙基硫代-β-D-半乳糖苷)(终浓度为1.0mmol/L)诱导表达4小时后制备蛋白电泳样品。11%聚丙烯酰胺凝胶电泳结果表明重组蛋白成功表达,得到抗猫杯状病毒(FCV)蛋白表达菌株。
实施例6:纯化猫杯状病毒(FCV)蛋白
接种重组蛋白表达菌株至LB液体培养基,加氨苄青霉素至终浓度为50μg/mL,37℃恒温摇床培养8小时后,用含50μg/mL氨苄青霉素的LB液体培养基将该菌按1:100比例稀释后,分装至细菌培养瓶,置37℃恒温摇床培养至OD600=0.8,加诱导剂IPTG(异丙基硫代-β-D-半乳糖苷)至终浓度为1.0mmol/L,继续培养诱导4小时。离心收集菌体后,4度低温超声破菌,低温离心后取上清通过镍琼脂糖亲和层析柱,经洗涤、洗脱最终得到纯化抗猫杯状病毒(FCV)蛋白。
实施例7:单链抗体scfv噬菌体库构建
取4-6周龄雌性Balb/c小鼠,基础免疫每只小鼠皮下多点注射弗氏完全佐剂乳化的100μg重组猫杯状病蛋白,共400μl/只。20天后进行第二次加强免疫,方法为取80μg重组猫杯状病蛋白用弗氏不完全佐剂乳化,共400μl/只,皮下多点注射。第三次加强免疫在15天以后,方法与第二次加强免疫相同。20天后,取120μg重组猫杯状病抗原腹腔加强注射,于72小时后,眼眶取血,并处死小鼠,取其脾脏用鼠脾脏淋巴细胞分离试剂盒(天津市灏洋生物制品科技有限责任公司)分离鼠脾脏淋巴细胞。用RNA提取试剂盒(天根生化科技有限公司)从分离的淋巴细胞中提取总RNA,用反转录试剂盒(Takara)反转录合成cDNA,用鼠源单链抗体scfv通用简并引物扩增重链可变区以及轻链可变区基因,PCR产物分别进行1%琼脂糖凝胶电泳,并分别切胶回收目的基因,回收的目的基因通过overlap PCR链接成scfv,PCR产物进行1%琼脂糖凝胶电泳,并切胶回收目的基因经NotI和SfiI酶切后使用T4连接酶和pCANTAB5e(北京宝科维食安生物技术有限公司)载体按一定比例于4℃连接12小时后,连接产物经胶回收试剂盒回收以去除里面的酶及缓冲物质,回收产物用细菌电转化仪(biorad)分多次电转入大肠杆菌TG1电转感受态,并涂布于含氨苄青霉素抗性(50μg/mL)及2%葡萄糖的2×YT-AG平板,于30℃恒温培养12小时之后,取适量的2×YT培养基用无菌玻璃棒将平板上的菌落全部刮取下来并收集菌体悬液,此为构建好的噬菌体抗体库。
实施例8:单链抗体scfv的淘选及筛选
从噬菌体抗体库中去取一定量的菌液接种到2×YT-AG培养液中使OD600为0.3。37℃,250rpm振荡1h左右,使OD600达0.5后加入辅助噬菌体M13K07超感染,感染比例为M13K07/TG1=20:1。37℃,250rpm振荡1h后3300g,4℃离心10min沉淀细菌,小心移弃上清。重悬细菌到含氨苄青霉素抗性(50μg/mL)及氨苄青霉素抗性(50μg/mL)的2×YT-AK培养基中,30℃,250rpm振荡培养过夜。次日,10800g,4℃离心20min沉淀细菌。上清移入干净离心管中,加入1/5体积的PEG/NaCl,混合后冰浴2h。10800g,4℃离心20min沉淀细胞,小心移弃上清,扣干,将沉淀重悬于PBS中,用0.45μm膜过滤去除细菌碎片用于淘选步骤。将纯化的重组猫杯状病抗原用包被液稀释至8μg/ml包被免疫管(Thermo),每个免疫管4ml,4℃过夜包被。次日,弃去包被液和未吸附的抗原,无菌PBST洗涤3次,每个免疫管加入封闭液5ml,37℃孵育2h。弃去封闭液,无菌PBST洗涤3次后将经PEG沉淀获得的噬菌体加入到免疫管中,每个免疫管加入4ml,37℃孵育1h。弃去免疫管中的液体,用无菌PBST洗涤10次再用无菌PBS洗涤10次后加入1ml 100mM三乙胺将结合的噬菌体洗脱下来,再立即加入500μl 1M Tris-HCl,pH 7.4进行中和。将中和后的噬菌体加入到一定量处于对数生长期的TG1大肠杆菌中进行超感染,此为第一轮淘选富集过程。经过3轮淘选,猫杯状病特异性scfv得以富集。将最后一轮洗脱中和后的噬菌体侵染TG1大肠杆菌后涂布于2×YT-AG平板上,于30℃恒温培养12小时之后随机挑取400-600个单克隆菌落于96孔深孔板中,用2×YT-AG培养基37℃,250rpm振荡2h后加入一定量M13K07辅助噬菌体进行超感染,37℃,250rpm振荡1h后离心去掉上清加入含氨苄青霉素抗性(50μg/mL)及氨苄青霉素抗性(50μg/mL)的2×YT-AK培养基30℃,250rpm过夜培养。第二天进行单克隆ELISA筛选,筛选步骤如下:
包被:用包被液稀释猫杯状病重组蛋白至终浓度为1μg/mL,100μL/孔加入酶标板(深圳金灿华实业有限公司),4℃过夜后通过DEM-3型洗板机(中山大学达安基因股份有限公司)用洗涤液洗涤1次;
封闭:以200μL/孔加入封闭液,37℃封闭2h,通过洗板机用洗涤液洗涤1次;
加样:加过夜诱导表达的细菌培养上清及对照血清,100μL/孔,37℃孵育1h,通过洗板机用洗涤液洗涤3次;
加酶标抗体:以100μL/孔加入新鲜稀释兔抗M13噬菌体HRP酶标二抗(购自北京义翘神州生物技术有限公司),37℃孵育30分钟后,通过洗板机用洗涤液洗涤4次;
加显色液:每孔加显色液A和显色液B各50μL,37℃避光显色10分钟;
终止反应:以50μL/孔加入2M H2SO4;
结果判定:在酶标仪上,于450nm处,空白孔校零后读取OD值。以免疫小鼠血清作为阳性对照。结果显示有14个阳性克隆OD值较高,经测序得到5株scfv序列,分别为3C5,6B1,2D8,5E2,7H4相关溶液配方如下:
包被液:Na2CO3 1.5g,NaHCO3 2.9g,加ddH2O定容至1000mL(pH9.6)。
封闭液:Na2HPO4·12H2O 2.68g,NaH2PO4·2H2O 0.39g,NaCl 8.5g,20g牛血清白蛋白,加ddH2O定容至1000mL(pH7.4)。
洗涤液:Na2HPO4·12H2O 2.68g,NaH2PO4·2H2O 0.39g,NaCl 8.5g,Tween-200.5mL,加ddH2O定容至1000mL(pH7.4)。
显色液A:200mg TMB溶于100mL无水乙醇,加ddH2O定容至1000mL。
显色液B:柠檬酸2.1g,Na2HPO4·12H2O 71g,加ddH2O定容至1000mL。
使用时:1mL显色液A+1mL显色液B+0.4μL 30% H2O2
终止液:2M H2SO4,21.7mL浓H2SO4加ddH2O定容至1000mL。
实施例9:真核表达载体构建及HEK293F细胞瞬转表达和纯化
将5株猫杯状病单链抗体scfv序列分别构建成完整鼠IgG1抗体序列,即将scfv中重链可变区与轻链可变区通过PCR分别与鼠IgG1重链恒定区和轻链恒定区桥接,再分别插入到pcDNA3.1(德国Novagen公司)质粒中。分别通过PEI将构建好的重链质粒与轻链质粒共转染HEK293F细胞,37℃,5%二氧化碳,120rpm细胞摇床表达7天后离心沉淀,收集上清过0.45μm滤器。用50mL平衡缓冲液PBS(pH7.4)平衡琼脂糖亲和介质Protein A层析柱(南京金斯瑞生物科技有限公司)至电脑核酸蛋白检测仪(上海沪西分析仪器厂有限公司)显示吸光度为0。上清上样后加PBS洗涤至吸光度为0,然后用0.1M甘氨酸(pH3.0)洗脱,收集流出液并加入500mM Tris-HCl(pH8.5)缓冲液中和至pH 7.0左右,得到纯化的单克隆抗体3C5,6B1,2D8,5E2,7H4
实施例10:Eu3+标记单抗的制备
取1mg纯化后的单克隆抗体于0.05mol/L碳酸盐缓冲液(pH9.6)中4℃透析3次,加入1mg DTPA(二乙基三胺五乙酸)立即混匀,室温反应1h,加入200μL Eucl3(33mmol/L),室温反应1h后用10m mol/L PBS(pH7.4)于4℃过夜透析。以上述方法分别对单抗3C5,6B1,2D8,5E2,7H4进行Eu3+标记。相关溶液配方如下:
碳酸盐缓冲液(pH9.6):Na2CO3 1.5g,NaHCO3 2.9g,加双蒸水定容至1000mL。PBS缓冲液(pH7.4):KH2PO4 0.29g,Na2HPO4·12H2O 2.9g,NaCl 8.2g,加双蒸水定容到1000mL。
实施例11:配对单抗筛选
5株单克隆抗体(3C5,6B1,2D8,5E2,7H4)分别经包被液稀释后(终浓度为1μg/mL),以100μL/孔加入酶标板(无锡国盛生物工程有限公司),4℃包被12小时后通过DEM-3型洗板机(中山大学达安基因股份有限公司)用洗涤液洗涤2次;加入封闭液,150μL/孔,37℃封闭1小时,洗板机洗板1次;取猫杯状病毒(FCV)感染的猫眼鼻口分泌物及正常猫眼鼻口分泌物于稀释液中溶解,100μL/孔,室温震荡孵育30分钟后,洗涤液洗涤5次;加实施例10制备得到的Eu3+标记单抗,100μL/孔,室温震荡孵育30分钟后,洗涤液洗涤5次;增强液100μL/孔,室温震荡5分钟,置于时间分辨检测仪(上海新波生物技术有限公司)读值。相关溶液配方如下:
包被液:Na2CO31.5g,NaHCO3 2.9g,加双蒸水定容至1000mL(pH9.6)。
封闭液:Na2HPO4.12H2O 2.68g,NaH2PO4.2H2O 0.39g,NaCl 8.5g,20g牛血清白蛋白,加双蒸水定容至1000mL(pH7.4)。
洗涤液:Na2HPO4.12H2O 2.68g,NaH2PO4.2H2O 0.39g,NaCl 8.5g,Tween-20 0.5mL,加双蒸水定容至1000mL(pH7.4)。
增强液:冰醋酸6mL,Triton X-100 1mL,TOPO(Tri-Octyl Phosphine Oxide正三辛基氧膦)50μmol,β-NTA(N(CH2COOH)3氨三乙酸)15μmol,用0.1mol/L的邻苯二甲酸氢钾调pH至3.2,加双蒸水定容至1000mL。
以上述方法正交检测各包被单抗与铕标单抗配对,求P/N值(阳性标本检测均值与阴性标本检测均值比值),见表1。
通过上表可知,3C5单抗包被与5E2-Eu配对检测猫杯状病毒(FCV)蛋白为最佳组合。
SEQ ID NO1:抗猫杯状病毒(FCV)蛋白特异性单链抗体scfv-3C5轻链可变区氨基酸序列;
SEQ ID NO2:抗猫杯状病毒(FCV)蛋白特异性单链抗体scfv-3C5重链可变区氨基酸序列;
SEQ ID NO3:抗猫杯状病毒(FCV)蛋白特异性单链抗体scfv-5E2轻链可变区氨基酸序列;
SEQ ID NO4:抗猫杯状病毒(FCV)蛋白特异性单链抗体scfv-5E2重链可变区氨基酸序列;
SEQ ID NO5:抗猫杯状病毒(FCV)蛋白特异性单链抗体scfv-3C5轻链可变区核苷酸序列;
SEQ ID NO6:抗猫杯状病毒(FCV)蛋白特异性单链抗体scfv-3C5重链可变区核苷酸序列;
SEQ ID NO7:抗猫杯状病毒(FCV)蛋白特异性单链抗体scfv-5E2轻链可变区核苷酸序列;
SEQ ID NO8:抗猫杯状病毒(FCV)蛋白特异性单链抗体scfv-5E2重链可变区核苷酸序列;
Claims (9)
1.猫杯状病毒(FCV)蛋白特异性单链抗体scfv-3C5,包括轻链和重链,其特征在于:
所述轻链可变区的氨基酸序列如SEQ ID NO.1所示;
所述重链可变区的氨基酸序列如SEQ ID NO.2所示。
2.猫杯状病毒(FCV)蛋白特异性单链抗体scfv-5E2,包括轻链和重链,其特征在于:
所述轻链可变区的氨基酸序列如SEQ ID NO.3所示;
所述重链可变区的氨基酸序列如SEQ ID NO.4所示。
3.一种编码权利要求1所述的猫杯状病毒(FCV)蛋白特异性单链抗体scfv-3C5的基因,其特征在于:
编码轻链可变区的核苷酸序列如SEQ ID NO.5所示;
编码重链可变区的核苷酸序列如SEQ ID NO.6所示。
4.一种编码权利要求2所述的抗猫杯状病毒(FCV)蛋白特异性单链抗体scFv-5E2的基因,其特征在于:
编码轻链可变区的核苷酸序列如SEQ ID NO.7所示;
编码重链可变区的核苷酸序列如SEQ ID NO.8所示。
5.一种质粒载体,其特征在于:该质粒载体含有权利要求3所述的轻链可变区核苷酸序列。
6.一种质粒载体,其特征在于:该质粒载体含有权利要求3所述的重链可变区核苷酸序列。
7.一种质粒载体,其特征在于:该质粒载体含有权利要求4所述的轻链可变区核苷酸序列。
8.一种质粒载体,其特征在于:该质粒载体含有权利要求4所述的重链可变区核苷酸序列。
9.权利要求5、6、7、8任意一项所述的质粒载体用于真核表达猫杯状病毒(FCV)单克隆抗体,其特征在于,包括:
(a)通过PCR将权利要求3、4中所述轻链和重链核苷酸序列分别与鼠IgG1轻链恒定区和重链恒定区核苷酸序列桥接后酶切,并分别连接质粒载体,构建真核细胞表达载体;
(b)将步骤(a)中真核表达载体转染至HEK293F细胞表达得到抗猫杯状病毒(FCV)蛋白单克隆抗体;
(c)纯化单抗并分别标记铕离子(Eu3+),通过正交实验确定最佳单抗配对组合。
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