CN1936001A - 鹅B淋巴细胞刺激因子cDNA及其克隆方法和重组应用 - Google Patents
鹅B淋巴细胞刺激因子cDNA及其克隆方法和重组应用 Download PDFInfo
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- CN1936001A CN1936001A CN200610096737.3A CN200610096737A CN1936001A CN 1936001 A CN1936001 A CN 1936001A CN 200610096737 A CN200610096737 A CN 200610096737A CN 1936001 A CN1936001 A CN 1936001A
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Abstract
鹅B淋巴细胞刺激因子(goose BAFF)cDNA及其克隆方法和重组应用,涉及生物基因工程领域。鹅B淋巴细胞刺激因子的基因,具有SEQ ID NO.1和SEQ ID NO.2所示的序列。其克隆方法如下:根据B淋巴细胞刺激因子的保守序列设计引物;从鹅脾脏提取总RNA;通过RT-PCR方法,一步扩增出全长cDNA序列,克隆入pMD18-T载体,挑取阳性克隆进行测序。所述鹅B淋巴细胞刺激因子的cDNA可通过现有基因工程方法,生产重组goose BAFF,作为鹅类免疫增强剂。
Description
技术领域
本发明涉及生物基因工程领域,具体涉及鹅B淋巴细胞刺激因子cDNA及其克隆、表达技术。
背景技术
人B淋巴细胞刺激因子(hBAFF)是1999年新发现的一种与人体免疫调控密切相关的肿瘤坏死因子家族成员,主要在外周血单核细胞、脾脏、淋巴结和骨髓中表达。hBAFF具有很强的B细胞趋化性,体外它作为B细胞增殖和分化的共刺激因子,能诱导活化的B细胞分泌大量的IgG、IgA、IgM等,对于休眠B细胞,hBAFF虽不能独立激活使之进入细胞周期循环,但通过上调细胞表面的抗凋亡蛋白Bcl-2、Bcl-XL的表达,延长休眠B细胞的寿命。体内它可以促进脾脏内T1-B细胞向T2-B细胞以及成熟B细胞的发育。hBAFF的正常表达是GC形成、抗体亲和力成熟、记忆性B细胞形成及B细胞正常发育的必须条件。BAFF-/-小鼠的次级淋巴器官囊外及过渡区B细胞完全缺失。hBAFF刺激B细胞增殖、分化并分泌抗体,成熟的B细胞及其产生的抗体构成了机体抵御感染和抑制肿瘤滋生的关键组分。由于hBAFF的免疫增强特异活性,自从被发现以来就显示了巨大的临床应用前景。2000年11月,美国FDA已正式批准重组人hBAFF进入人体临床试验,用于治疗普通变异免疫缺乏症(CVID)患者。
根据GenBank、EMBL和DDBJ国际三大主要核酸序列数据库搜索结果得知,目前只有人、鼠、鸡、鸭的BAFF cDNA已被克隆,并分别已在GenBank数据库中申请了序列号,序列号分别是AF116456、AF119383、AF506010及DQ445092。鹅B淋巴细胞刺激因子(gBAFF)cDNA还未被克隆出来,关于鹅BAFF基因的研究在整个国内外还处于完全空白状态。鹅是十分重要的禽类,由于B淋巴细胞刺激因子(BAFF)具有较强的免疫增强能力,基因工程鹅BAFF蛋白有可能开发成一种能增强鹅类免疫能力的生物制剂,用于体质弱、免疫功能低下的病鹅,增加鹅类抗病毒能力,尤其是在禽流感对人类已构成威胁的今天。近年来,随着对细胞因子研究的不断深入和分子生物学技术的发展,大量的禽类细胞因子得以克隆及重组表达,许多细胞因子基因工程药物面市,给治疗禽类疾病提供了新的手段,同时创造了巨大的经济和社会效益,因此,重组鹅BAFF蛋白具有潜在的巨大市场应用价值。同时,B淋巴细胞刺激因子(BAFF)是新发现的免疫系统重要调控因子,对鹅BAFF的研究有助于探索鹅类的免疫调控机制,推动其免疫系统研究的发展。
发明内容
本发明针对现有技术的不足,提供一种从鹅提取的B淋巴细胞刺激因子cDNA,并提供鹅B淋巴细胞刺激因子cDNA的克隆方法及重组技术。
本发明从鹅中克隆到B淋巴细胞刺激因子cDNA,它具有SEQ ID NO.1所示的序列。
鹅B淋巴细胞刺激因子,它具有SEQ ID NO.2所示的序列。
本发明的鹅B淋巴细胞刺激因子cDNA的克隆方法如下:
(1)根据鸡,鸭B淋巴细胞刺激因子的保守序列设计引物:
正义寡核苷酸引物gBAFF1:5’-GGAGGAAAGAGCAGCGATG-3’
反义寡核苷酸引物gBAFF2:5’-GCAGTAAGCATAACAACAGAATC-3’
(2)从鹅脾脏提取总RNA,
(3)通过RT-PCR方法,以上述引物gBAFF1及gBAFF2为特异性引物,扩增出一段cDNA序列,克隆入pMD18-T载体,并对其碱基序列进行测定,
上述鹅B淋巴细胞刺激因子cDNA的克隆方法具体操作如下:
(1)根据B淋巴细胞刺激因子全长cDNA两段高保守区域设计同源克隆的正义寡核苷酸引物gBAFF1(5’-GGAGGAAAGAGCAGCGATG-3’)与反义寡核苷酸引物gBAFF2(5’-GCAGTAAGCATAACAACAGAATC-3’),
(2)应用RNA抽提试剂TRIzol(Invitrogen公司),按照操作手册,提取出鹅脾脏细胞的总RNA,
(3)应用RT-PCR方法,以上述gBAFF1及gBAFF2为特异性引物,扩增出一段cDNA序列,全长为914bp,克隆入pMD18-T载体,并对其碱基序列进行测定。RT-PCR的反应条件为:以总RNA为模板,在50μl反应体系中,加入5×RT-PCR反应缓冲液10μl、25mM MgSO42μl、10mM dNTP混合物1μl、20μM的上下游引物各2.5μl、AMV逆转录酶(Takara公司)1μl、Tf1 DNA聚合酶1μl及RNA模板2μl,补水至50μl。RT-PCR循环参数为:48℃逆转录45min,94℃变性2min,再进行29个PCR循环(94℃变性30s,56℃退火30s,72℃延伸30s),最后于72℃延伸7min。RT-PCR产物用1%的琼脂糖凝胶电泳分离后,用胶回收试剂盒回收约914bp的DNA条带,克隆入pMD18-T载体,挑取8个阳性克隆进行碱基序列测定。
将所得序列与GenBank数据库序列进行相似性搜索,发现与已知的鸭、鸡、人、鼠BAFF序列相似率最高,分别是98%、92%、55%、44%,可以确定该序列是鹅B淋巴细胞刺激因子(gBAFF)的全长cDNA,其开放阅读框如SEQ ID NO.1所示。
对该基因的进一步研究表明:该基因主要表达在鹅类免疫器官中,包括法氏腔上囊、脾脏及胸腺。其中法氏腔上囊和脾脏表达水平较高,胸腺次之;该基因的重组融合蛋白具有gBAFF的高活性功能;该基因编码的蛋白对鹅B淋巴细胞具有明显的促存活/增殖效应;对人及鼠的B淋巴细胞都具有促存活/增殖的生物学功能;充分表明本发明克隆得到的基因就是鹅B淋巴细胞刺激因子(gBAFF)的cDNA。
上述鹅B淋巴细胞刺激因子(gBAFF)的cDNA的重组应用,通过现有基因工程方法,生产重组goose BAFF,作为鹅类免疫增强剂。
所说的鹅B淋巴细胞刺激因子cDNA通过现有基因工程方法,转入鹅体内,增加其免疫力,在饲养中应用。
附图说明
图1是goose BAFF全长cDNA的克隆过程示意图;箭头代表引物位置;全长cDNA共914bp。
图2是goose BAFF全长cDNA碱基序列及推测编码氨基酸序列。
图3是goose BAFF与鸭、鸡、人、鼠BAFF全长氨基酸序列同源性比较图。其中阴影代表保守的氨基酸;箭头所指代表弗林蛋白酶识别位点,切割后的C端为BAFF的胞外可溶功能区。
图4是goose BAFF mRNA在各组织中的表达水平分析图。β-actin作为内参。
图5是融合蛋白His-gsBAFF在BL21(DE3)中的诱导表达,Ni+柱纯化的SGS-PAGE鉴定及鼠抗His6-tag的western-blotting鉴定结果,A图中条带1是经IPTG诱导4.5小时后的全菌蛋白,2是未经IPTG诱导的全菌蛋白,3是经Ni+柱纯化后目的蛋白;4是鼠抗His6-tag单抗的western-blotting鉴定结果,5是兔抗His-gsBAFF多抗的western-blotting鉴定结果。
图6是本发明克隆的基因对鹅B淋巴细胞的促存活作用结果示意图。图A是说明不同浓度的His-gsBAFF相对于对照His-TRAIL在作用鹅B淋巴细胞48小时后,对鹅B淋巴细胞可见显著的促存活作用,且呈剂量依赖效应;图B是说明固定浓度的His-gsBAFF相对于对照His-TRAIL分别在作用鹅B淋巴细胞24/48/72小时后的鹅B淋巴细胞存活数量比较情况,PMA为阳性对照,His-gsBAFF的兔多抗能中和His-gsBAFF的生物学活性。
图7是goose BAFF、duck BAFF和chicken BAFF之间存在功能交叉反应。图A是说明不同浓度的His-gsBAFF对鸡、鸭、鹅B淋巴细胞的促存活作用;图B是说明不同浓度的His-gsBAFF对鸡、鸭、鹅B淋巴细胞的促存活作用;图C是说明不同浓度的His-csBAFF对鸡、鸭、鹅B淋巴细胞的促存活作用
具体实施方式
在本发明中所使用的术语,除非有另外说明,一般具有本领域普通技术人员通常理解的含义。
下面结合具体的制备实施例和应用实施例,并参照数据进一步详细地描述本发明。应理解,这些实施例只是为了举例说明本发明,而非以任何方式限制本发明的范围。
在以下的实施例中,未详细描述的各种过程和方法是本领域中公知的常规方法。所用到的引物,均在首次出现时标明,其后所用相同引物,均以首次标明的内容相同。
实施例1:
鹅(Anser cygnoides),人工饲养。
(1)引物设计:应用ClustalX软件对已克隆出的人、鼠、鸡、鸭B淋巴细胞刺激因子全长cDNA序列进行同源性比对分析,精心选取出两段高保守区域用来设计同源克隆的正义寡核苷酸引物gBAFF1(5’-GGAGGAAAGAGCAGCGATG-3’)与反义寡核苷酸引物gBAFF2(5’-GCAGTAAGCATAACAACAGAATC-3’)。
(2)提取总RNA:应用RNA抽提试剂TRIzol(Invitrogen公司)按照其操作手册提取出约0.5克鹅脾脏细胞的总RNA,并通过甲醛变性琼脂糖凝胶电泳鉴定其质量和纯度,紫外分光光度计测定其浓度。
(3)应用RT-PCR方法,以上述gBAFF1及gBAFF2为特异性引物,扩增出goose BAFFcDNA的一段序列,全长为914bp,克隆入pMD18-T载体,并对其碱基序列进行测定。RT-PCR的反应条件为:以总RNA为模板,在50μl反应体系中,加入5×RT-PCR反应缓冲液10μl、25mM MgSO42μl、10mM dNTP混合物1μl、20μM的上下游引物各2.5μl、AMV逆转录酶(Takara公司)1μl、Tf1 DNA聚合酶1μl及RNA模板2μl,补水至50μl。RT-PCR循环参数为:48℃逆转录45min,94℃变性2min,再进行29个PCR循环(94℃变性30s,56℃退火30s,72℃延伸30s),最后于72℃延伸7min。RT-PCR产物用1%的琼脂糖凝胶电泳分离后,用胶回收试剂盒回收约914bp的DNA条带,克隆入pMD18-T载体,经含Amp抗生素(50mg/ml)的琼脂糖平板筛选转化子,挑取出8个阳性克隆送往上海英骏测序公司进行碱基序列测定,得到如图2所示的goose BAFF全长cDNA碱基序列及推测编码氨基酸序列。
(6)同源检索:将所得序列向
http://www.ncbi.nlm.nih.gov/BLAST/提交克隆得到的cDNA序列在GenBank、EMBL和DDBJ国际三大主要核酸序列数据库进行相似性搜索及同源性分析,如图3所示,发现与已知的鸭(GenBank登录号DQ445092)、鸡(GenBank登录号AF506010)、人(GenBank登录号AF116456)、鼠(GenBank登录号AF119383)BAFF氨基酸序列相似性最高(不包括其他种属BAFF的预测氨基酸序列),分别是98%、92%、55%、44%,可以确定该序列是鹅B淋巴细胞刺激因子(gBAFF)的全长cDNA。并且其开放阅读框具有SEQ ID NO.1序列,碥码的蛋白的氨基酸序列如SEQ ID NO.2所示。
实施例2:
goose BAFF 基因在各组织的表达水平分析
采用实施例1中的提取RNA的方法,分别提取了鹅肝(1iver)、肾(kidney)、胸腺(thymus)、腔上囊(bursa)、脾(spleen)、心(heat)及脑(brain)的总RNA,运用半定量RT-PCR法对goose BAFF基因在各组织的表达水平进行了研究,结果如图4所示,goose BAFF基因主要表达在鹅类免疫器官中,包括法氏腔上囊、脾脏及胸腺;其中法氏腔上囊和脾脏表达水平较高,胸腺次之。试验中鹅β-actin作为内参,采用的引物为gβ-actin1(5’-ATCGTCACCAACTGGGACGACATGG-3’)和gβ-actin1(5’-TCTCCTGCTCGAAGTCCAGGGCGAC-3’)。RT-PCR体系如实施例1。
可溶性goose BAFF重组载体的构建及其在大肠杆菌中的诱导表达
以实施例1得到的goose BAFF全长cDNA为模板,设计引物gsBAFF1(5’-TCAGGACATATGTCTGTCGTCCACACGGAAG-3’(Nde I))及gsBAFF1(5’-ACTGAGGGATCCTCAGAAGAGTCTGACGGCACCA-3’(BamH I))扩增出goose BAFF cDNA胞外可溶区段(gsBAFF),引物分别引入酶切位点Nde I及BamH I,亚克隆入pET-28a载体,构建成重组载体pET-28a-gsBAFF。将此重组载体转入大肠杆菌表达菌株BL21(DE3),胞浆内可高可溶性表达出目的蛋白His-gsBAFF,此融合蛋白经促存活与增殖试验(图6,7),活性检测证实具有gsBAFF的高活性功能。
重组目的蛋白的的Ni+亲和纯化
IPTG诱导含有重组载体pET-28a-gsBAFF的大肠杆菌表达菌株BL21(DE3)4.5小时后,4℃收菌,冰上超声破碎(工作10s,暂停10s,共99次)表达菌体,4℃,13,000×g,20min离心超声破碎液后,弃沉淀,留上清过Ni+-NTA柱。简要过程是:3体积Binding Buffer平衡Ni+-NTA柱后,手动上样含可溶性重组蛋白的上清,6体积WashBuffer(60mM咪唑,0.5M NaCl,20mM Tris-HCl pH7.9)洗去未结合上Ni+-NTA柱的杂蛋白,最后6体积Elute Buffer(1M咪唑,0.5M NaCl,20mM Tris-HCl pH7.9)洗脱目的蛋白,PBS透析除盐,以SGS-PAGE及毛细管电泳分析纯度并以紫外分光光度计检测蛋白浓度,如图5(A,条带3)所示,表达得到的目的蛋白经Ni+柱亲和纯化得到纯度达95%的活性目的蛋白。
western印迹分析
Western-blot中所用一抗为羊抗His6单抗和兔抗His-gsBAFF多抗,二抗为辣根过氧化酶标记的鼠抗羊和羊抗兔IgG。其简要步骤是:将诱导的产物先行SGS-PAGE,然后以浸入式法将蛋白电转移至硝酸纤维素膜(NC膜)上,用记号笔标记蛋白marker各条带,然而依次经5%脱脂奶粉室温封闭1h,与一抗孵育1h,与HRP标记的二抗IgG孵育1h,每步完成后均严格洗膜,最后加TMB避光显色。结果如图5所示在目的蛋白位置出现了特异性条带。
goose BAFF对鹅法氏腔上囊B淋巴细胞促存活作用实验
采用淋巴细胞分离液常规无菌分离鹅法氏腔上囊淋巴细胞(约98%为B淋巴细胞),用RPMI1640调节细胞密度至5×107个/mL.将B细胞悬液加入96孔培养板,每孔100μl。向每孔内加入不同浓度的His-gsBAFF/His-TRAIL,37℃,5%CO2培养72h后每孔加入10μl 5mg/mLMTT,37℃,5%CO2继续培养5h,加入100μl SGS-HCl溶解沉淀物,37℃过夜,测定各孔OD570值实验设三复孔,取其平均值,并计算标准误差。在体外通过MTT细胞毒试验检测goose BAFF对鹅B淋巴细胞的促存活作用,如图6所示,结果表明本发明克隆得到的goose BAFF对鹅B淋巴细胞具有明显的促存活效应,与鸭、鸡、人、鼠BAFF的生物学功能相同,且呈现剂量依赖效应,即当His-gsBAFF为3μg/ml时goose BAFF的促增殖作用最强。同时,固定浓度的His-gsBAFF相对于对照His-TRAIL分别在作用鹅B淋巴细胞24/48/72小时后,可观察到72小时时对鹅B淋巴细胞的促存活效应最明显。
goose BAFF、duck BAFF和chicken BAFF之间存在功能交叉反应
采用淋巴细胞分离液从鸡、鸭、鹅法氏囊中无菌分离鸡、鸭、鹅B淋巴细胞,在体外通过MTT细胞毒试验检测goose BAFF、duck BAFF和chicken BAFF对鸡、鸭、鹅B淋巴细胞的促存活,方法同上,结果如图7所示,表明goose BAFF、duck BAFF和chicken BAFF之间存在功能交叉反应,且呈现剂量依赖效应,饱和浓度分别为3μg/ml,4μg/ml和3μg/ml,进一步证实了BAFF在生物体内生物学功能的保守性。
实施例3:
将实施例1获得的鹅B淋巴细胞刺激因子cDNA通过现有基因工程方法,生产重组goose BAFF,作为鹅类免疫增强剂。
实施例4:
将实施例1获得的鹅B淋巴细胞刺激因子cDNA通过现有基因工程方法,转入鹅体内,增加其免疫力,在饲养中应用。
按照本发明的方法可以通过现有基因工程技术,修饰鹅B淋巴细胞刺激因子基因并用于所述研究和生产。
SEQUENCE LISTING
<110>南京师范大学
<120>鹅B淋巴细胞刺激因子cDNA及其克隆方法和重组应用
<160>2
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<211>867
<212>cDNA
<213>鹅(Anser cygnoides)
<400>1
atgaaatccg tggactgtgt gcacgtcatc caacagaagg ataccgcctc ctctccctct 60
gccccccccg gtgctgcttc aggcaccacg ggactttttt ctgtcacatt cctgtggctt 120
gcaatgctcc tgtcctcttg tcttgcagca gtgtctcttt accatgttct caccctgaaa 180
acagagctag aagctctgcg ccatgagctg atctacaggg tccaggcaag gtctccgcta 240
gagcggccag ccgtctcccc tgatgacgag aaagcgggtg cccctgtttc ttccttcctg 300
caagcctctg cagctggtgc caggcaggag aacaagcttc ctggccctag cccagctgaa 360
agtttccgaa acgaaatctc gaatgggagc agaaacaggg ggagaaggtc tgtcgtccac 420
acggaagaaa cagtgctgca ggcctgcttg caactgatcg ctgacagcaa aagtgatatc 480
caacagaaag atgattcaag cattgtccct tggcttctga gctttaaacg tggaacagct 540
ctggaagaac atggaaataa aatagtgatc aaagaaacag gatatttttt catatatggc 600
caggttttat atacagatac aacatttgct atgggacatc taatacagag gaagaaggcc 660
catgtgtttg gtgatgatct gagcttggtg acattatttc gctgcataca aaatatgcca 720
cagtcttatc cgaataattc ttgctatact gctggcattg caaaattaga agaaggggac 780
gaacttcaac ttacaatacc acgaagacga gccaaaatat ccttggatgg cgatggtact 840
ttttttggtg ccgtcagact cttctga 867
<210>2
<211>288
<212>PRT
<213>鹅(Anser cygnoides)
<400>2
Met Lys Ser Val Asp Cys Val His Val Ile Gln Gln Lys Asp Thr
1 5 10 15
Ala Ser Ser Pro Ser Ala Pro Pro Gly Ala Ala Ser Gly Thr Thr
20 25 30
Gly Leu Phe Ser Val Thr Phe Leu Trp Leu Ala Met Leu Leu Ser
35 40 45
Ser Cys Leu Ala Ala Val Ser Leu Tyr His Val Leu Thr Leu Lys
50 55 60
Thr Glu Leu Glu Ala Leu Arg His Glu Leu Ile Tyr Arg Val Gln
65 70 75
Ala Arg Ser Pro Leu Glu Arg Pro Ala Val Ser Pro Asp Asp Gln
80 85 90
Lys Ala Gly Ala Pro Val Ser Ser Phe Leu Gln Ala Ser Ala Ala
95 100 105
Gly Ala Arg Gln Glu Asn Lys Leu Pro Gly Pro Ser Pro Ala Glu
110 115 120
Ser Phe Arg Asn Glu Ile Ser Asn Gly Ser Arg Asn Arg Gly Arg
125 130 135
Arg Ser Val Val His Thr Glu Glu Thr Val Leu Gln Ala Cys Leu
140 145 150
Gln Leu Ile Ala Asp Ser Lys Ser Asp Ile Gln Gln Lys Asp Asp
155 160 165
Ser Ser Ile Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Thr Ala
170 175 180
Leu Glu Glu His Gly Asn Lys Ile Val Ile Lys Glu Thr Gly Tyr
185 190 195
Phe Phe Ile Tyr Gly Gln Val Leu Tyr Thr Asp Thr Thr Phe Ala
200 205 210
Met Gly His Leu Ile Gln Arg Lys Lys Ala His Val Phe Gly Asp
215 220 225
Asp Leu Ser Leu Val Thr Leu Phe Arg Cys Ile Gln Asn Met Pro
230 235 240
Gln Ser Tyr Pro Asn Asn Ser Cys Thr Thr Ala Gly Ile Ala Lys
245 250 255
Leu Glu Glu Gly Asp Glu Leu Gln Leu Thr Ile Pro Arg Arg Arg
260 265 270
Ala Lys Ile Ser Leu Asp Gly Asp Gly Thr Phe Phe Gly Ala Val
275 280 285
Arg Leu Phe
288
Claims (4)
1、鹅B淋巴细胞刺激因子cDNA,其特征在于,具有SEQ ID NO.1所示的序列。
2、一种权利要求1所述的鹅B淋巴细胞刺激因子cDNA的克隆方法如下:
(1)根据B淋巴细胞刺激因子的保守序列设计引物:
正义寡核苷酸引物gBAFF1:5’-GGAGGAAAGAGCAGCGATG-3’,
反义寡核苷酸引物gBAFF2:5’-GCAGTAAGCATAACAACAGAATC-3’
(2)从鹅脾脏提取总RNA,
(3)通过RT-PCR方法,以上述引物gBAFF1及gBAFF2为特异性引物,扩增出一段cDNA序列,克隆入pMD18-T载体,挑取阳性克隆进行碱基序列测定。
3、一种权利要求1所述的鹅B淋巴细胞刺激因子cDNA的重组应用,是通过现有基因工程方法,生产重组鹅B淋巴细胞刺激因子,作为鹅类免疫增强剂。
4、鹅B淋巴细胞刺激因子,其特征在于,具有SEQ ID NO.2所示的序列。
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Cited By (4)
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CN101870975A (zh) * | 2010-06-13 | 2010-10-27 | 南京师范大学 | 鹧鸪B淋巴细胞刺激因子cDNA及其克隆方法和重组应用 |
CN102115748A (zh) * | 2010-12-21 | 2011-07-06 | 南京师范大学 | 斑马鱼B淋巴细胞刺激因子cDNA及其克隆方法和重组应用 |
CN102121010A (zh) * | 2010-12-21 | 2011-07-13 | 南京师范大学 | 暗纹东方鲀B淋巴细胞刺激因子cDNA及其克隆方法和重组应用 |
CN109091671A (zh) * | 2018-09-26 | 2018-12-28 | 中国农业科学院北京畜牧兽医研究所 | 鹅特异性寡脱氧核苷酸免疫佐剂及其应用 |
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Cited By (5)
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CN101870975A (zh) * | 2010-06-13 | 2010-10-27 | 南京师范大学 | 鹧鸪B淋巴细胞刺激因子cDNA及其克隆方法和重组应用 |
CN102115748A (zh) * | 2010-12-21 | 2011-07-06 | 南京师范大学 | 斑马鱼B淋巴细胞刺激因子cDNA及其克隆方法和重组应用 |
CN102121010A (zh) * | 2010-12-21 | 2011-07-13 | 南京师范大学 | 暗纹东方鲀B淋巴细胞刺激因子cDNA及其克隆方法和重组应用 |
CN102121010B (zh) * | 2010-12-21 | 2012-07-25 | 南京师范大学 | 暗纹东方鲀B淋巴细胞刺激因子cDNA及其克隆方法和重组应用 |
CN109091671A (zh) * | 2018-09-26 | 2018-12-28 | 中国农业科学院北京畜牧兽医研究所 | 鹅特异性寡脱氧核苷酸免疫佐剂及其应用 |
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