CN106008708A - 一种人乙型肝炎病毒x蛋白的单克隆抗体及用途 - Google Patents
一种人乙型肝炎病毒x蛋白的单克隆抗体及用途 Download PDFInfo
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Abstract
本发明涉及生物医药和基因工程领域,提供了一种针对乙型肝炎病毒(HBV)X蛋白(HBx)的单克隆抗体(命名为KY01)及其轻链可变区和重链可变区氨基酸序列,以及制备HBx单克隆抗体KY01的方法和可生产KY01抗体的杂交瘤细胞系。所述的KY01抗体能够特异性识别乙型肝炎病毒已知8种基因型中7种基因型(A、B、C、D、E、F和H)编码的X蛋白,并与其中第61-120位氨基酸区段特异结合,与HBx蛋白其它部位、HBV其它蛋白、FLAG标签和人蛋白组无特异性反应。本发明的KY01抗体可用于针对HBx蛋白的免疫检测和特异性细胞靶向,为研发HBV感染及其它相关疾病的诊断和治疗方法提供支持。
Description
技术领域
本发明涉及生物医药和基因工程领域,具体涉及一种可特异性识别7个亚型人乙型肝炎病毒(HBV)X蛋白(HBx)的单克隆抗体及其在制备诊断和治疗HBV感染与相关疾病如肝细胞癌(HCC)的药物中的应用。
背景技术
有报道公开了由HBV感染引起的乙型肝炎以肝脏炎性病变为主,并可并发多器官损害。HBV感染慢性化为慢性乙型肝炎(CHB)后,还可引发肝纤维化、肝硬化、原发性肝细胞癌(HCC)和肝衰竭,导致死亡。近年来,随着HBV疫苗的成熟和预防接种的推广,新发HBV感染率显著下降,但全球仍然有3.5亿左右HBV慢性携带者,每年约1百万患者死于HBV感染相关疾病(Lavanchy 2004)。中国属HBV感染高发区,约有1.2亿慢性携带者和3000万慢性乙型肝炎患者(Liu,Si et al.2002)。
有研究公开,乙型肝炎病毒X蛋白(HBx)由HBV的X基因编码,包括154个氨基酸残基(Tiollais,Pourcel et al.1985)。已知的8种HBV基因型(A型-H型)均编码相同长度的HBx蛋白,不同基因型HBx之间的氨基酸序列同源性可低至70%左右。培养细胞中的研究表明,HBx在细胞内易发生降解,半衰期约30分钟左右,因此通常在细胞内以较低水平存在(Schek,Bartenschlager et al.1991)。
已有研究表明HBx是HBV诱发肝癌的重要因素之一。HBx不仅存在于HBV相关HCC病人的肿瘤组织中,也存在于非肿瘤组织中(Moriarty,Alexander et al.1985;Wang,London et al.1991;Diamantis,McGandy et al.1992)。对HBV感染患者肝脏和血清中HBx蛋白或相应抗体进行连续检测,对于研究急/慢性HBV感染患者的病程进展,特别是HBV相关HCC的早期发现、发展观察和疗效评价,具有现实的临床意义。此外,由于HBx仅在HBV感染细胞中表达,且不具备分泌信号肽,故通常情况基本不进入患者血液,因此,HBx同时也是靶向性治疗慢性乙型肝炎和HBV相关HCC的重要靶标分子。
针对HBx的基础和临床研究与应用需要具有高特异性和广泛反应性的HBx单克隆抗体,在此基础上,本申请的发明人拟开发HBx蛋白及抗体的检测系统、针对表达HBx细胞的靶向性工具等诊断和治疗工具,并进而通过人源化、单链化等分子改造得到药学性质更为优良的抗体分子,尤其是提供一种人乙型肝炎病毒X蛋白的单克隆抗体及用途。
发明内容
本发明的目的是提供能够特异性识别多种亚型HBx蛋白的单克隆抗体,具体涉及一种人乙型肝炎病毒X蛋白的单克隆抗体,包括其轻链和重链可变区及互补决定区的氨基酸序列和核苷酸序列,为构建性质更优的人源化抗体、单链抗体等基因工程抗体提供支持。
本发明的另一目的是提供检测多种亚型HBx蛋白及其相应抗体的方法。
本发明通过以下技术方案实现:
能够识别除G型以外7种亚型HBV编码的HBx蛋白的单克隆抗体,包含重链可变区和轻链可变区,所述重链可变区中的3个互补决定区(CDR)氨基酸序列分别为:
CDR1:Gly Tyr Thr Phe Thr Ser Tyr;
CDR2:Asp Pro Ser Asn Ser Glu;
CDR3:Asp Gly Ala Tyr;
所述轻链可变区中的3个CDR氨基酸序列分别为:
CDR1:Arg Ala Ser Glu Ser Val Asp Ser Tyr Gly Asn Ser Phe Ile His;
CDR2:Arg Ala Ser Asn Leu Glu Ser;
CDR3:Gln Gln Ser Asn Glu Asp Pro Arg Thr。
所述的HBx蛋白单克隆抗体,编码重链可变区的基因序列如SEQ ID NO.1所示,编码轻链可变区的基因序列如SEQ ID NO.2所示。
所述的HBx蛋白单克隆抗体,重链可变区氨基酸序列如SEQ ID NO.3所示,轻链可变区序列氨基酸序列如SEQ ID NO.4所示。
本发明还提供了一种杂交瘤细胞株,命名为Anti-HBx KY01,已于2014年4月2日保藏,保藏单位:中国典型培养物保藏中心,保藏单位地址:中国.武汉.武汉大学(湖北省武汉市武昌区武汉大学保藏中心(武汉大学第一附属小学对面)),其保藏编号为CCTCC NO:C201446,所述细胞株能够分泌产生所述的HBx蛋白单克隆抗体。
本发明中,根据所述的HBx蛋白单克隆抗体CDR序列,通过基因工程方法构建的相应的单链抗体、人(鼠)嵌合抗体或人源化抗体。
本发明还提供所述的HBx蛋白单克隆抗体在用于检测人乙型肝炎病毒X蛋白中的应用。
本发明还提供所述的HBx蛋白单克隆抗体在制备治疗人乙型肝炎病毒感染及与之相关肝脏疾病的药物中的应用。
目前公开文献中虽有关于HBx单克隆抗体制备和应用的报道,市场上亦有仅限于研究应用的HBx单克隆或多克隆抗体在售,但上述HBx抗体均缺乏针对不同亚型HBx反应性的公开、有效数据,其中的单克隆抗体亦未公开其氨基酸及核苷酸编码序列,无法进行进一步的基因工程改造。
本发明提供了针对HBx的小鼠单克隆抗体KY01能够特异性识别HBV已知8个基因型中除G型外的7个常见基因型编码的HBx蛋白;所述KY01能够在Western blot检测中识别变性的HBx蛋白,能够在ELISA检测中识别未变性的HBx蛋白,能够在免疫荧光检测中识别细胞内的天然形式HBx蛋白;不仅如此,KY01还能够在免疫组化法中检测到HBV相关肝癌病人癌细胞中的HBx抗原;实验显示KY01在临床样本中HBx蛋白及其抗体检测中具有广泛的应用价值,可用于针对HBx蛋白的诊断和治疗方法的研发。
本发明还公开了KY01的重链和轻链可变区序列,为通过基因工程方法改造KY01获得具有更优特性的重组抗体用于诊断和治疗建立了基础。
与现有技术相比,本发明具有以下有益的技术效果:
1、本发明提供的HBx单克隆抗体(命名为KY01),在免疫印迹和免疫荧光检测方法中可特异性识别已知8种HBV基因型中除G型以外的7种基因型(A-F及H型)所编码的HBx蛋白,所述抗体特异性地与HBx蛋白第61-120位氨基酸区段特异结合,与HBx蛋白其它部位、HBV其它蛋白、Flag和人蛋白组无特异性反应。
2、本发明克隆并测定了HBx单克隆抗体KY01的轻链可变区和重链可变区的基因序列及对应的氨基酸序列,证实其序列唯一性。
3、通过分析KY01的轻链可变区和重链可变区氨基酸序列,得到了轻链和重链互补决定区CDR1-3的氨基酸序列,为构建具有更优药学及其它特性的基因工程抗体提供支持。
附图说明
图1,Anti-HBx KY01杂交瘤细胞培养上清纯化得到的单克隆抗体KY01的纯度鉴定和滴度检测结果,
其中显示,A:纯化的单克隆抗体KY01经还原性SDS-PAGE电泳(分离胶浓度12%)后,考马斯蓝染色,两条主要条带分别为抗体重链(高分子量条带)和轻链(低分子量条带);B:96孔板中包被原核表达的HBx抗原并以脱脂奶粉封闭后,加入梯度稀释的纯化单克隆抗体KY01孵育,以ELISA方法确定纯化抗体的滴度为1:2500000。阳性阈值设置为0.3(横线显示)。
图2,单克隆抗体KY01在Western blot检测中对8个基因型(A-H)HBx的识别情况,其中显示,A-H共8个亚型的HBx编码序列克隆入带有FLAG标签的真核表达载体后转染HEK293T细胞,细胞裂解液中的FLAG-HBx融合蛋白通过Western blot方法分别以FLAG抗体(上图)和HBx抗体KY01(下图)检测。
图3,单克隆抗体KY01在免疫荧光检测中对8个基因型(A-H)HBx的识别情况,
其中显示,A-H共8个亚型的HBx编码序列克隆入带有FLAG标签的真核表达载体后转染HEK293T细胞,使用HBx抗体KY01作为一抗进行免疫荧光检测(红色),同时以DAPI(蓝色)对细胞核染色;pc代表空载体质粒转染对照。
图4显示了单克隆抗体KY01在Western blot检测中特异性识别HBx第61-120氨基酸残基区段,其中左图显示通过定点突变方法由野生型全长HBx基因出发构建的系列缺失突变体Xm1-Xm5,克隆入带有FLAG标签的真核表达载体后转染HEK293T细胞,细胞裂解液中的FLAG-HBx融合蛋白通过Western blot方法分别以FLAG抗体(右上图)和HBx抗体KY01(右下图)检测。
图5显示单克隆抗体KY01在免疫组织化学方法检测HBV相关肝癌患者肝组织中HBx抗原表达中的应用结果,其中,4例HBV相关肝癌患者组织病理切片中有3例显示明显的HBx表达(褐色产物沉积)。
图6显示单克隆抗体KY01重链可变区(VH)和轻链可变区(VL)脱氧核糖核苷酸序列,其中,重链可变区CDR1、CDR2和CDR3以及轻链可变区CDR1、CDR2和CDR3编码序列以下划线方式标示。
图7显示单克隆抗体KY01重链可变区和轻链可变区氨基酸序列,其中重链可变区CDR1、CDR2和CDR3以及轻链可变区CDR1、CDR2和CDR3编码序列以下划线方式标示。
具体实施方式
下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。除非另有描述,本发明的实施将采用免疫学、分子生物学、微生物学和重组DNA等常规技术,这些均是本领域技术人员所知的。这些技术在下列文献中有完整的描述:例如,Sambrook《分子克隆实验指南》第2版(1989);《DNA克隆》第I和II卷(D.N.Glover编辑,1985);《寡核苷酸合成》(M.J.Gait编辑,1984);《核酸杂交》(B.D.Hames和S.J.Higgins编辑,1984);《蛋白质纯化:原理和实践》第2版(Springer-Verlag,N.Y.),以及《实验免疫学手册》I-IV卷(D.C.Weir和C.C.Blackwell编辑,1986)。或者,可按照试剂生产商所提供的说明书进行。
实施例1:制备针对HBx的小鼠单克隆抗体
首先将B基因型HBx(GenBank NO.AF100309.1)编码序列克隆入原核表达载体,在大肠杆菌中重组表达并纯化重组HBx蛋白,以50微克HBx蛋白免疫BALB/c小鼠两次后采血, 通过ELISA方法检测血清中HBx抗体的产生,血清抗体滴度达到峰值后处死小鼠,制备脾细胞悬液与小鼠骨髓瘤SP2/0细胞通过常规PEG处理法诱导融合,融合细胞接种于96孔培养板,在重氮丝氨酸/次黄嘌呤选择培养基中培养;待杂交瘤细胞生长形成可见集落,以免疫原HBx蛋白包板通过ELISA方法测定上清液中的HBx抗体活性,对阳性集落进一步通过有限稀释法进行单克隆化,至抗体表达分泌性状稳定,最终获得一株稳定分泌高滴度单克隆HBx抗体的杂交瘤细胞株,命名为Anti-HBx KY01,其所分泌的单克隆抗体命名为KY01;
单克隆抗体KY01的大量制备按常规方法进行,如通过腹水法得到的小鼠腹水经硫酸铵沉淀浓缩后,利用偶联蛋白G的琼脂糖凝胶介质进行亲和纯化,纯化产物以SDS-PAGE法鉴定纯度,结果如图1左图所示,纯化的KY01呈现清晰的轻链和重链条带,纯度超过95%;纯化产物梯度稀释后以免疫原HBx蛋白包板通过ELISA法测定抗体滴度(效价),结果如图1右图所示,抗体滴度可达到1:2.5E6;经抗体亚型鉴定,单克隆抗体KY01为小鼠IgG1亚类,κ型轻链。
实施例2:单克隆抗体KY01特异识别7种亚型的HBx蛋白
KY01由B基因型HBx蛋白免疫小鼠制备得到,而已知HBV存在至少8种基因型A-H,各基因型HBx蛋白氨基酸序列的同源性可低至70%左右;将8种基因型的HBx蛋白与FLAG标签融合后在HEK293T细胞中进行表达,细胞裂解液在变性条件下电泳后分别以FLAG抗体和KY01进行Western blot检测,结果如图2所示,表明单克隆抗体KY01可以识别除G型以外的其余7种亚型HBx;此外,利用KY01对表达FLAG-HBx融合蛋白的HEK293T进行免疫荧光检测,结果如图3所示,表明KY01在非变性条件下同样可以识别除G型以外的其余7种亚型HBx;G型是临床上较为少见的HBV基因型,且通常与其它基因型共同存在,因此,所述的KY01可以识别绝大多数临床常见亚型HBV编码的HBx蛋白。
实施例3:单克隆抗体KY01特异识别HBx蛋白的第61-120位氨基酸残基
为确定KY01识别的HBx区段(表位),通过定点突变方法由野生型全长HBx基因出发构建缺失HBx不同区段的系列缺失突变体Xm1-Xm5,克隆入带有FLAG标签的真核表达载体后转染HEK293T细胞,细胞裂解液中的FLAG-HBx融合蛋白通过Western blot方法分别以FLAG抗体和HBx抗体KY01检测,结果如图4所示,表明KY01识别的HBx抗原表位位于第61-120位氨基酸残基构成的片段之内。
实施例4:单克隆抗体KY01用于HBx蛋白的免疫组织化学检测
采用切除的与HBV感染相关的肝癌患者的肝癌组织制作病理切片,以所述的KY01抗体通过免疫组织化学方法检测肝癌组织中HBx蛋白的表达,结果显示,KY01在3例标本检测到了明确的HBx蛋白信号(如图5所示)。
实施例5:单克隆抗体KY01轻链和重链可变区基因的克隆
培养的杂交瘤细胞Anti-HBx KY01使用常规TRIZol法抽提RNA并反转录为cDNA,以下述PCR引物对分别扩增KY01轻链和重链可变区:
轻链可变区引物对:
上游引物:5'-GGATACAGTTGGTGCAGCATC-3';
下游引物:5'-GACATTGTGCTGACCCAATCT-3';
重链可变区引物:
上游引物:5'-GGCCAGTGGATAGTCAGATGGGGGTGTCGTTTTGGC-3';
下游引物:5'-GAGGTCCAGCTGCAGCAGTC-3';
扩增得到的轻链可变区和重链可变区PCR产物片段克隆入TA克隆载体pMD19(TAKARA公司),经质粒测序后获得KY01重链可变区核苷酸序列SEC ID NO.1和轻链可变区核苷酸序列SEC ID NO.2(如图6所示);在核苷酸序列基础上,通过遗传密码翻译获得KY01重链可变区氨基酸序列SEC ID NO.3和轻链可变区核苷酸序列SEC ID NO.4(如图7所示);
KY01重链可变区氨基酸序列使用abYsis分析平台、以CHOTHIA分区原则(Chothia et al.,1992)分析确定其中3个CDR位置,得到其氨基酸序列分别为SEC ID NO.5、SEC ID NO.6、SEC ID NO.7(如图7所示);
KY01轻链可变区氨基酸序列使用abYsis分析平台、以CHOTHIA分区原则分析确定其中3个CDR位置,得到其氨基酸序列分别为SEC ID NO.8、SEC ID NO.9、SEC ID NO.10(如图7所示);
KY01可变区核苷酸及氨基酸序列与GenBank收录的已公开序列比对(Blastn及Blastp方法),未发现完全相同序列;与已公开序列的部分同源性主要发生在已公开小鼠抗体可变区序列中的骨架区,而CDR区未发现同源的已公开序列,表明本发明的KY01具有序列上的唯一性。
本发明实验结果显示了针对HBx的小鼠单克隆抗体KY01能够特异性识别HBV已知8个基因型中除G型外的7个常见基因型编码的HBx蛋白;KY01能够在Western blot检测中 识别变性的HBx蛋白,能够在ELISA检测中识别未变性的HBx蛋白,能够在免疫荧光检测中识别细胞内的天然形式HBx蛋白;以及,KY01还能够在免疫组化法中检测到HBV相关肝癌病人癌细胞中的HBx抗原,表明本发明所述的KY01在临床样本中HBx蛋白及其抗体检测中具有广泛的应用价值,可用于针对HBx蛋白的诊断和治疗方法的研发。
SEQUENCE
LISTING
<110>复旦大学,上海科垣生物医药科技有限公司
<120> 一种人乙型肝炎病毒X蛋白的单克隆抗体及用途
<130>20150325
<160>10
<170>PatentIn version 3.1
<210> 1
<211> 339
<212> DNA
<213> 小鼠
<400> 1
gaggtccagc tgcagcagtc tgggcctgag ctggtgaggc ctggggcttc agtgaagatg 60
tcctgcaagg cttcaggcta taccttcacc agttactgga tgcactggtt gaaacagagg 120
cctggacaag gccttgagtg gattggcatg attgatcctt ccaatagtga aactaggtta 180
aatcagaagt tcaaggacaa ggccacattg aatgttgaca aatcctccaa cacagcctac 240
atgcagctca gcagcctgac atctgaggac tctgcagtct attactgtgc ctacgacgga 300
gcttactggg gccaagggac tctggtcact gtctctgaa 339
<210> 2
<211> 339
<212> DNA
<213> 小鼠
<400> 2
gacattgtgc tgacccaatc tccagcttct ttggctgtgt ctctagggca gagggccacc 60
atatcctgca gagccagtga aagtgttgat agttatggca atagttttat acactggtac 120
cagcagaaac caggacagcc acccaaactc ctcatctatc gtgcatccaa cctagaatct 180
gggatccctg ccaggttcag tggcagtggg tctaggacag acttcaccct caccattaat 240
cctgtggagg ctgatgatgt tgcaacctat ttctgtcagc aaagtaatga ggatcctcgg 300
acgttcggtg gaggcaccaa gctggaaatc aaacgggct 339
<210> 3
<211> 113
<212> PRT
<213> 小鼠
<400> 3
Glu Val Gln Leu Gln
Gln Ser Gly Pro Glu Leu Val Arg
Pro Gly Ala
1 5 10 15
Ser Val Lys
Met Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Ser Tyr
20 25 30
Trp Met His Trp Leu Lys Gln
Arg Pro Gly Gln Gly Leu
Glu Trp Ile
35 40 45
Gly Met Ile
Asp Pro Ser Asn Ser Glu Thr Arg Leu
Asn Gln Lys Phe
50
55 60
Lys Asp Lys
Ala Thr Leu Asn Val Asp Lys Ser Ser Asn Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser
Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Tyr Asp Gly Ala Tyr Trp Gly Gln Gly
Thr Leu Val Thr Val Ser
100 105 110
Glu
<210> 4
<211> 113
<212> PRT
<213> 小鼠
<400> 4
Asp Ile Val Leu Thr Gln
Ser Pro Ala Ser Leu Ala Val Ser Leu
Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Tyr
20 25 30
Gly Asn Ser Phe Ile His Trp Tyr Gln Gln
Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly
Ile Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Arg Thr
Asp Phe Thr Leu Thr Ile Asn
65 70 75 80
Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Phe Cys
Gln Gln Ser Asn
85 90 95
Glu Asp Pro Arg Thr Phe
Gly Gly Gly
Thr Lys Leu Glu Ile Lys Arg
100 105 110
Ala
<210> 5
<211> 7
<212> PRT
<213> 小鼠
<400> 5
Gly Tyr Thr Phe Thr
Ser Tyr
1 5
<210> 6
<211> 6
<212> PRT
<213> 小鼠
<400> 6
Asp Pro Ser Asn Ser Glu
1 5
<210> 7
<211> 4
<212> PRT
<213> 小鼠
<400> 7
Asp Gly Ala Tyr
1
<210> 8
<211> 15
<212> PRT
<213> 小鼠
<400> 8
Arg Ala Ser Glu Ser Val Asp Ser Tyr Gly Asn Ser Phe Ile His
1 5 10 15
<210> 9
<211> 7
<212> PRT
<213> 小鼠
<400> 9
Arg Ala Ser Asn Leu Glu
Ser
1 5
<210> 10
<211> 9
<212> PRT
<213> 小鼠
<400> 10
Gln Gln Ser Asn Glu
Asp Pro Arg Thr
1 5
Claims (7)
1.一种抗人乙型肝炎病毒X蛋白单克隆抗体,包含重链可变区和轻链可变区,其特征在于:
a)所述重链可变区中的3个互补决定区(CDR)氨基酸序列分别为:
CDR1:Gly Tyr Thr Phe Thr Ser Tyr;
CDR2:Asp Pro Ser Asn Ser Glu;
CDR3:Asp Gly Ala Tyr;和
b)所述轻链可变区中的3个互补决定区(CDR)氨基酸序列分别为:
CDR1:Arg Ala Ser Glu Ser Val Asp Ser Tyr Gly Asn Ser Phe Ile His;
CDR2:Arg Ala Ser Asn Leu Glu Ser;
CDR3:Gln Gln Ser Asn Glu Asp Pro Arg Thr。
2.如权利要求1所述的单克隆抗体,其特征在于:其中,
a)编码重链可变区的基因序列如SEQ ID NO.1所示,编码轻链可变区的基因序列如SEQID NO.2所示;或
b)重链可变区氨基酸序列如SEQ ID NO.3所示,轻链可变区序列氨基酸序列如SEQ IDNO.4所示。
3.一株分泌抗人乙型肝炎病毒X蛋白小鼠单克隆抗体的杂交瘤细胞株,命名为Anti-HBxKY01,保藏在中国典型培养物保藏中心,其保藏编号为CCTCC NO:C201446。
4.如权利要求2所述的单克隆抗体,特征在于,所述的抗体由权利要求3所述的杂交瘤细胞株分泌产生。
5.如权利要求1所述的抗人乙型肝炎病毒X蛋白单克隆抗体,其特征在于所述的抗体是单链抗体、人(鼠)嵌合抗体或人源化抗体。
6.权利要求1、2、4或5所述的单克隆抗体在用于检测人乙型肝炎病毒X蛋白中的应用。
7.权利要求1、2、4或5所述的单克隆抗体在制备治疗人乙型肝炎病毒感染及相关肝脏疾病药物中的应用。
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Cited By (3)
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---|---|---|---|---|
CN107132357A (zh) * | 2017-03-23 | 2017-09-05 | 山东大学 | 一种逆转乙型肝炎病毒慢性感染的抗Tim‑3抗体和α‑galcer的组合及应用 |
CN110343714A (zh) * | 2018-04-08 | 2019-10-18 | 复旦大学 | 一种HBV中HBx蛋白核心区段的可溶化和纯化及单克隆抗体 |
CN116162153A (zh) * | 2022-09-01 | 2023-05-26 | 复旦大学附属中山医院 | 一种乙肝病毒表面抗原的单克隆抗体及其应用 |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102030824A (zh) * | 2009-09-30 | 2011-04-27 | 王虹 | 一种乙型肝炎病毒x蛋白单克隆抗体及用途 |
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---|---|---|---|---|
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Non-Patent Citations (2)
Title |
---|
刘湘: "乙型肝炎病毒X蛋白的原核表达及单克隆抗体的制备和鉴定", 《重庆医科大学硕士学位论文》》 * |
谢南等: "乙型肝炎病毒X蛋白定量试验与临床研究", 《中国生物工程杂志》 * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107132357A (zh) * | 2017-03-23 | 2017-09-05 | 山东大学 | 一种逆转乙型肝炎病毒慢性感染的抗Tim‑3抗体和α‑galcer的组合及应用 |
CN107132357B (zh) * | 2017-03-23 | 2019-11-01 | 山东大学 | 一种逆转乙型肝炎病毒慢性感染的抗Tim-3抗体和α-galcer的组合及应用 |
CN110343714A (zh) * | 2018-04-08 | 2019-10-18 | 复旦大学 | 一种HBV中HBx蛋白核心区段的可溶化和纯化及单克隆抗体 |
CN116162153A (zh) * | 2022-09-01 | 2023-05-26 | 复旦大学附属中山医院 | 一种乙肝病毒表面抗原的单克隆抗体及其应用 |
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