CN1534092A - 一种含人源导向溶栓抗体基因的大肠杆菌及其分泌产物 - Google Patents
一种含人源导向溶栓抗体基因的大肠杆菌及其分泌产物 Download PDFInfo
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Abstract
本发明涉及一种含人源抗D-二聚体的特异性导向溶栓抗体基因的大肠杆菌及其分泌产物。其特征在于通过半套式PCR方法将人抗体的重链基因和轻链基因连入噬菌体质粒pComb3H形成重组噬菌体质粒pComb3H-HL,该重组噬菌体质粒在辅助噬菌体VCSM13的超感染下构建成噬菌体抗体组合文库;经ELISA方法筛选出抗人纤维蛋白降解产物D-二聚体的特异株。因此,该抗体具有对血栓降解物D-二聚体特异的免疫性,能够与溶栓药物组成导向溶栓剂,在治疗血管栓塞性疾病方面具有广泛的临床应用价值。
Description
技术领域:
本发明涉及一种含人源抗D-二聚体的特异性导向溶栓抗体基因的大肠杆菌及其分泌产物。
背景技术:
溶解血栓,恢复血流通畅是治疗急性心肌梗死和脑梗塞等血管栓塞性疾病的最有效的方法。组织型纤溶酶原激活物(t-PA)、尿激酶(Urokinase,UK)是临床常用的高效溶栓药。但人体其它部位组织细胞存在较多的t-PA结合受体,可降低循环中t-PA的浓度,使其治疗作用减弱。而加大用药剂量,不仅治疗成本提高,而且大剂量的t-PA或UK亦能激活循环血中的纤溶酶原,降解循环血中的纤维蛋白、α2-抗纤溶酶、凝血因子V、VIII及血小板GP IIb/IIIa受体复合物等凝血物质,造成全身性出血倾向。近年来也有不少文献资料报道导向溶栓,但其疗效并不令人满意,主要存在以下几个方面的缺陷:(1)在血栓靶向标志物的选择上多以纤维蛋白为主,而纤维蛋白β-链N端的特异性抗原决定区在溶栓早期随纤维蛋白的聚合而部分遮蔽或丧失,降低了纤维蛋白单体的靶向性。(2)制备单克隆抗体所采用的细胞杂交瘤技术,抗体生产的稳定性差。(3)杂交瘤技术生产的单克隆抗体多为鼠源性,分子量大,对人具有免疫原性,降低溶栓效果,甚至可导致严重的过敏反应。(4)导向制剂多采用化学交联方法连接特异性单克隆抗体与溶栓剂,其化学交联的化学修饰作用可降低抗体与血栓部位的结合,并导致溶栓剂活性降低。因此,研究一种新型的溶栓药物的导向剂就十分必要。
D-二聚体(D-Dimer,DD)在血栓形成过程中一直与血栓的核心相连,其抗原性不被遮蔽,是已交联纤维蛋白降解形成的γ-γ链为主的纤维蛋白特异性降解产物,为纤维蛋白降解产物中的最小片段,在正常人血中基本无此物,是唯一直接反映凝血酶和纤溶酶生成的理想指标,当纤溶作用增强时血中才会大量出现,其含量的增高表明机体的凝血和纤溶系统的双重相继激活。因此D-二聚体可作为体内高凝状态和继发纤溶亢进的分子标志物之一。
噬菌体展示技术(phage display)的出现得益于用PCR的方法人为地设计一组引物扩增全套免疫球蛋白可变区基因,以及在大肠杆菌中表达并分泌出具有识别、结合抗原能力的免疫球蛋白分子的成功。20世纪80年代以来,噬菌体展示技术经Dullbeclo提出并在随后的时间里被广泛应用,成为一项制备特异性抗体的新方法,是一种基因表达筛选技术。它提供了一条从抗体库中分离目的基因的有效手段,并可将目的基因克隆在特定表达载体中,使外源基因产物与外壳蛋白融合,展示在丝状噬菌体表面。可以绕过杂交瘤途径,甚至不经过免疫制备具有诊断和治疗价值的人源单克隆抗体,在新型药物设计、研制导向转移系统方面,具有广阔的应用前景。
pComb3H是专用于表达抗体基因的质粒。噬菌体pComb3H质粒有氨苄青霉素抗性基因。pComb3H通常在大肠杆菌XL1-Blue中表达。噬菌体的Fd-PIII融合蛋白基因被翻译成蛋白后通过pelB先导序列被导入细菌的细胞膜间隙,重链Fd通过与PIII融合镶嵌在膜上,而轻链通过ompA被引导入膜间隙,而细胞膜间隙又有良好的氧化环境,使重链和轻链得以形成良好稳定的二硫键而组成有功能的Fab,由于噬菌体感染细菌后其外壳蛋白的合成也是通过上述类似途径,致使功能性的Fab抗体可组装在噬菌体表面。
技术内容:
为了解决溶栓药物的导向性问题,本发明提供:一种产生人源抗D-二聚体的特异性导向溶栓抗体的大肠杆菌,其特征在于通过半套式PCR方法将人抗体的重链基因和轻链基因连入噬菌体质粒pComb3H形成重组噬菌体质粒pComb3H-HL,重组噬菌体质粒pComb3H-HL在辅助噬菌体VCSM13的超感染下构建成噬菌体抗体组合文库;经ELISA方法筛选出抗人纤维蛋白降解产物D-二聚体的特异株。
为了获得人源导向溶栓抗体。首先经PCR扩增出人免疫球蛋白基因:分离人外周血中的淋巴细胞,用TRIZOL溶液提取细胞总RNA,用于cDNA的合成。扩增引物参照Kang等报道的序列加以改动进行第一次PCR,用4组Fd前导肽引物与8组重链可变区3’端引物配对组成32对引物,Kappa链可变区前导肽引物与Kappa链可变区3’端引物配对,Lambda链可变区前导肽引物与Lambda链可变区3’端引物配对。
第一次PCR引物:
●人源重链Fd引物
1)重链可变区前导肽引物
506-1 5’-ATG GAC TAA ACC TGG AGG (A/G)TC (C/T)TC T(G/T)C-3’
506-2 5’-ATG GAG (C/T)TT GGG CTG A(G/C)G TGG (G/C)TT T(C/T)T-3’
506-3 5’-ATG(A/G)A(A/C) (A/C)(A/T)A CT(G/T) TG(G/T) (A/T)(G/C/T)C (A/T)(C/T)(G/C)
CT(C/T)C(C/T)G-3’
2)重链可变区3’端引物
CA1 5’-AGT TGA
ACT AGT TGG GCA GGG CAC AGT CAC-3’
CE1 5’-GCT GAA
ACT AGT TTT GTC GAC CCA GTC TGT GGA-3’
CD1 5’-TGC CTT
ACT AGT CTC TGG CCA GCG GAA GAT-3’
CG4A 5’-GCA TGA
ACT AGT TGG GGG ACC ATA TTT GGA-3’
CG2A 5’-CTC GAC
ACT AGT TTT GCG CTC AAC TGT CTT-3’
CG3A 5’-TGT GTG
ACT AGT GTC ACC AAG TGG GGT TTT-3’
CG1Z 5’-GCA TGT
ACT AGT TTT GTC ACA AGA TTT GGG-3’
CM1 5’-GCT CAC
ACT AGT AGG CAG CTC AGG AAT CAC-3’
●人源轻链引物
3)Kappa链可变区前导肽引物
506-4 5’-ATG GAC ATG (AG)(AG)(AG) (AGT)(CT)C C(ACT)(AGC) G(CT)(GT) CA(GC) CTT-3’
4)Kappa链可变区3’端引物
Ck1Z 5’-GCG CCG
TCT AGA ACT AAC ACT CTC CTC TGT TGA AGC TCT TTG TGA CGG GCG ATC TCA G-3’
5)Lambda链可变区前导肽引物
507-1 5’-ATG (TG)CC TGG (AT)C(CT) CCT CTC (CT)T (CT) CT(CG) (AT)(CT)C-3’
6)Lambda链可变区3’端引物
CLZ 5’-CGC CG
T CTA GAA TTA TGA ACA TTC TGT AGG-3’
取第一次PCR产物作模板进行第二次扩增,用5组重链可变区5’端引物与8组重链可变区3’端引物配对组成40对重链引物,用2组Kappa链可变区5’端引物与Kappa链可变区3’端引物配对组成两对Kappa链引物,用8组Lambda链可变区5’端引物与Lambda链可变区3’端引物配对组成8对Lambda链引物。重链可变区5’端引物中含有XhoI酶切位点(CTCGAG),重链可变区3’端引物含有SpeI酶切位点(ACTAGT),轻链可变区5’端引物含有SacI酶切位点(GAGCTC),轻链可变区3’端引物含有XbaI酶切位点(TCTAGA)。
第二次PCR引物:
●人源重链Fd引物
1)重链可变区5’端引物
VH13A 5’-AG GTG CAG
CTC GAG (C/G)AG TCT GGG-3’
H58 5’-CAG GTA CAG CTG
CTC GAG TCA GGT CCA-3’
VH134F 5’-AG GTG CAG CTG
CTC GAG TC(T/G) GG-3’
VH4FG 5’-CAG GTG CAG CT(A/G)
CTC GAG TCT GG-3’
VH6A 5’-CAG GTA CAG
CTC GAG CAG TCA GG-3’
2)重链可变区3’端引物
CA1 5’-AGT TGA
ACT AGT TGG GCA GGG CAC AGT CAC-3’
CE1 5’-GCT GAA
ACT AGT TTT GTC GAC CCA GTC TGT GGA-3’
CD1 5’-TGC CTT
ACT AGT CTC TGG CCA GCG GAA GAT-3’
CG4A 5’-GCA TGA
ACT AGT TGG GGG ACC ATA TTT GGA-3’
CG2A 5’-CTC GAC
ACT AGT TTT GCG CTC AAC TGT CTT-3’
CG3A 5’-TGT GTG
ACT AGT GTC ACC AAG TGG GGT TTT-3’
CG1Z 5’-GCA TGT
ACT AGT TTT GTC ACA AGA TTT GGG-3’
CM1 5’-GCT CAC
ACT AGT AGG CAG CTC AGG AAT CAC-3’
●人源轻链引物
3)Kappa链可变区5’端引物
HK53 5’-GAT ATT
GAG CTC ACT CAG TCT CCA-3’
VK1S 5’-GAC ATC
GAG CTC ACC CAG TCT CC-3’
4)Kappa链可变区3’端引物
HK3 5’-GCG CCG
TCT AGA ACT AAC ACT CTC CCC TGT TGA AGC TCT TTG TGA CGG GCG ATC TCA G-3’
5)Lambda链可变区5’端引物
HL51 5’-AAT TTT
GAG CTC ACT CAG CCC CAC-3’
HL53 5’-TCT GTG
GAG CTC CAG CCG CCC TCA GTG-3’
HL56 5’-CAG TCT
GAG CTC ACT CAG GAG CCC-3’
VL5.7 5’-CAG (G/T)(C/T)T
GAG CTC AC(G/T) CA(A/G) CCG CCC-3’
VL1GP 5’-AAT TTT
GAG CTC ACT CAG CCC CCC TCT GTG TC-3’
VL2 5’-TCT G(A/C/T)(A/C/G)
GAG CTC CAG (C/G)(A/C)(C/G/T) (C/G)C(C/T) (G/T)(C/T)(A/C/T)
GTG-3’
VL5.8 5’-CAG(A/G/T)CT
GAG CTC ACGCAG(C/G)(A/C)G(C/T)C(C/T)TCC-3’
VL2.3 5’-TCT G(C/T)(C/G)
GAG CTC CAG CC(T/G)(C/G)CC TC(A/C)GTG-3’
6)Lambda链可变区3’端引物
HL3 5’-CGC CG
T CTA GAA CTA TGA ACA TTC TGT AGG-3’
含有人免疫球蛋白重链基因和轻链基因的噬菌体质粒pComb3H-HL的构建。轻链扩增产物经SacI-XbaI双酶切,pComb3H也经SacI-XbaI双酶切,T4DNA连接酶连接轻链基因和pComb3H,构成pComb3H-L。重链扩增产物经XhoI-SpeI双酶切,pComb3H-L也经XhoI-SpeI双酶切,用T4DNA连接酶连接,构成pComb3H-HL。
富集筛选抗人纤维蛋白降解产物D-二聚体的pComb3H-HL噬菌体株。用pH9.6的碳酸钠-碳酸氢钠溶液包被D-二聚体,噬菌体抗体库吸附在上,经吹打将吸附不上或吸附不紧的噬菌体抗体吸走,分别用pH2.2的甘氨酸-盐酸洗脱缓冲液和Tris溶液洗脱并中和吸附紧密的噬菌体抗体,加入大肠杆菌XL1-Blue孵育1小时,再加入辅助噬菌体VCSM13培养,利用噬菌体的可增殖性扩增对D-二聚体特异的噬菌体抗体。
用pComb3H-HL/XL1-Blue系统表达抗体。用SpeI-NheI双酶切将geneIII基因及其后的终止子切去,利用SpeI和NheI是同裂酶的关系,用连接酶将线性质粒连接成环,并在大肠杆菌XL1-Blue中表达,由于缺少了geneIII基因,不能表达PIII蛋白,噬菌体无法组装,只能表达出抗体的重链片段和轻链片段,这两个片段被引导肽引导入胞质周间装配成Fab。
经过ELISA和Western blotting验证,从噬菌体抗体库中筛选及在大肠杆菌中表达的抗体确对人纤维蛋白降解产物D-二聚体具有抗性和特异性。
上述含pComb3H-HL的大肠杆菌已交“中国典型培养物保藏中心(武汉)”保藏,保藏号CCTCC NO:M203009
综上所述,本发明获得了一种人源导向溶栓抗体,该抗体对人纤维蛋白降解产物D-二聚体具有特异性结合能力。因此,该抗体能与溶栓药物结合,将溶栓药物专一引导到血栓上,集中发挥溶栓药物的功效,避免了药物的无效释放,是一种高效的溶栓导向剂。具有极高的临床应用价值。
上述工程菌能分泌含以下人源抗体轻链氨基酸序列,其特征在于:
S V S E S P G K T V T I S C T
G S S G S I A S N Y V Q W Y R
Q R P G S A P T T V I Y E D N
Q R P S G V P D R F S G S I D
S S T N S A S L T I S G L K T
E D E A D Y Y C Q S Y D S S N
W V F G G G T K L T V L G Q P
K A A P S V T L F P P S S E E
L Q A N K A T L V C L I S D F
Y P G A V T V A W K A D S S P
V K A G V E T T T P S K Q S N
N K Y A A S S Y L S L T P E Q
W E S H K S Y S C Q V T H E G
S T V E K T V A
上述人源导向溶栓抗体的轻链的基因序列,其特征在于:
TCTGTGTCGGAGTCTCCGGGGAAGACGGTAACCATCTCCTGCACC
GGCAGCAGTGGCAGCATTGCCAGCAACTATGTGCAGTGGTACCGG
CAGCGCCCGGGCAGTGCCCCCACCACTGTGATCTATGAGGATAAC
CAAAGACCCTCTGGGGTCCCTGATCGGTTCTCTGGCTCCATCGAC
AGCTCCACCAACTCTGCCTCCCTCACCATCTCTGGACTGAAGACT
GAGGACGAGGCTGACTACTACTGTCAGTCTTATGATAGCAGCAAT
TGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCC
AAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAG
CTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTC
TACCCGGGAGCCGTGACGGTGGCCTGGAAGGCAGATAGCAGCCCC
GTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAAC
AACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAGCAG
TGGGAGTCCCACAAAAGCTACAGCTGCCAGGTCACGCATGAAGGG
AGCACCGTGGAGAAGACAGTGGCC
上述工程菌能分泌含以下人源抗体重链氨基酸序列,其特征在于:
E S G G G V V Q P G R S L R L
S C A A S G F T F S S Y A M S
W V R Q A P G K G L E W V S A
I S G S G G S T Y Y A D S V K
G R F T I S R D N S K N T L Y
L Q M D S L R A E D T A V Y Y
C A K P H G R Y Y D S S G Y R
T Y F D Y W G Q G T L V T V S
S G S A S A P T L F P L V S C
E N S P S D T S S V A V G C L
A Q D F L P D S I T F S W K Y
K N N S D I S S T R G F P S V
L R G G K Y A A T S Q V L L P
S K D V M Q G T D E H V V C K
V Q H P N G N K E K N V P L P
V I A E L P
上述人源导向溶栓抗体的重链的基因序列,其特征在于:
GAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTC
TCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGC
TGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCT
ATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAG
GGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTAT
CTGCAAATGGACAGCCTGAGAGGCGAGGACACGGCCGTATATTAC
TGTGCGAAGCCCCACGGGCGATACTATGATAGTAGTGGTTATCGA
ACCTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCC
TCAGGGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTCCTGT
GAGAATTCCCCGTCGGATACGAGCAGCGTGGCCGTTGGCTGCCTC
GCACAGGACTTCCTTCCCGACTCCATCACTTTCTCCTGGAAATAC
AAGAACAACTCTGACATCAGCAGCACCCGGGGCTTCCCATCAGTC
CTGAGAGGGGGCAAGTACGCAGCCACCTCACAGGTGCTGCTGCCT
TCCAAGGACGTCATGCAGGGCACAGACGAACACGTGGTGTGCAAA
GTCCAGCACCCCAACGGCAACAAAGAAAAGAACGTGCCTCTTCCA
GTGATTGCAGAGCTGCCT
附图说明:
图1重组质粒pComb3H-HL结构示意图
图2人Kappa链片段PCR电泳图
实施方案:本发明通过以下实施例进一步阐述。
淋巴细胞的分离纯化和细胞总RNA的提取
抽取人外周血,用淋巴细胞分离液分离淋巴细胞,按照常规方法提取细胞总RNA。用无RNAase的纯水重悬RNA,置于冰浴用于cDNA合成或置-70℃保存。(注:病人和正常人的血液分别各自混合,提取总RNA)
将上述从淋巴细胞提取的RNA5ul置于无RNAase的Eppendorf管中,组成如下体系:
Oliga(dT) 1μl
RNA 5μl
DEPC处理水 4μl
将Eppendorf管置于65℃5min,然后置冰浴。在用前将cDNA合成缓冲液振荡5s。制备以下反应体系:
5×cDNA合成缓冲液 4μl
0.1M DTT 1μl
RNAase抑制剂 1μl
DEPC处理水 1μl
10mM dNTP Mix 2μl
反转录酶THERMOSCRIPT RT 1μl
total 10μl
将上述反应混合液加到每个置冰浴的RNA引物混合管中,置50℃ 20~50分钟。置85℃5分钟终止反应。瞬时离心,加1μlRNAaseH于每个反应管中,37℃反应20分钟。马上用于PCR或-20℃长期保存。
半套式PCR扩增Fab抗体基因
分别采用病人与正常人的cDNA为模板,用上述第一次PCR引物扩增。程序如下:94℃变性5min后,加入TaqDNA聚合酶,再94℃变性1min,热降落55℃~45℃退火1min,72℃延伸1min,10个循环后,再94℃变性1min,48℃退火1min,72℃延伸1min,15个循环后,72℃延伸5min。
反应体系如下:
信号肽端引物 0.5μl
3’端引物 0.5μl
10×PCR缓冲液 2μl
dNTPs 1μl
去离子水 14μl
cDNA 1μl
Taq酶 1μl
Total 20μl
取上述制备的PCR产物为模板进行第二次PCR反应。PCR循环前先在94℃变性5min,然后加入TaqDNA聚合酶,再94℃变性1min,热降落62℃~50℃退火1min,72℃延伸1min,12个循环后,再94℃变性1min,54℃退火1min,72℃延伸1min,20个循环后,72℃延伸5min。
反应体系如下:
5’端引物 1μl
3’端引物 1μl
10×PCR缓冲液 5μl
dNTPs 1μl
去离子水 40μl
cDNA 1μl
Taq酶 1μl
Total 50μl
取PCR产物进行电泳分析,结果表明均获得~700bp的目的条带(图2所示为人抗体Lambda链片段PCR产物电泳图,“1~8”分别为引物对HL3/HL51、HL3/HL53、HL3/HL56、HL3/VL5.7、HL3/VL1GP、HL3/VL2、HL3/VL5.8和HL3/VL2.3的PCR产物,“9”为阴性对照,“10”为100bp Marker)
噬菌体抗体库的构建
将载体pComb3H DNA和经过纯化的轻链PCR产物分别用Xba I和Sac I酶切,T4DNA连接酶连接,16℃12~16小时。电转化感受态细菌XL1-Blue,于SOC培养基培养过夜。取1μl培养液分别稀释100倍、1000倍涂布于含有100μg/ml氨苄青霉素的LB平板,以检测转化效率。其余菌液转入10mlSB(含Amp20μg/ml)培养基,37℃振荡培养1小时。加入140ml SB(含有Amp100μg/ml),37℃振荡培养过夜。次日提取重组质粒pComb3H-L DNA,进
行以下操作:
1.鉴定插入效率。随机挑取20个克隆,用菌落PCR法鉴定重组率:在超净台中用灭菌牙签从37℃过夜的LB平板上随机挑取20个菌落,混入含已配好的20μlPCR反应体系中,94℃变性5min,再94℃30s,54℃40s,72℃50s,35个循环后72℃延伸10min。将PCR产物在新鲜的TAE缓冲液中用2%的琼脂糖凝胶电泳,扩增出680bp左右的片段者,为重组克隆。鉴定轻链重组率的引物:
5’端 5’-GGC TGG TTT CGC TAC CGT GGC CCA G-3’
3’端 5’-GCT GCC GTA GGC AAT AGG TAT TTC A-3’
鉴定重链重组率的引物:
5’端 5’-CTG CCC AAC CAG CCA TGG CCG AAG-3’
3’端 5’-CCT CCT GGC CGG CCT GGC CAC TAG-3’
2.重链基因的克隆及噬菌体抗体库的建立。将上述经过纯化的重链Fd PCR产物和含有轻链基因的pComb3H-L分别进行Xho I和Spe I酶切反应。连接、转化以及鉴定方法同上。得到重组质粒pComb3H-HL(图1)。
噬菌体抗体库的富集筛选
将D-二聚体用0.1mmol/l碳酸钠-碳酸氢钠(PH9.6)溶液稀释为10μg/ml,每孔50μl,4℃过夜。次日洗去板中未吸附抗原,用3%BSA-PBS 37℃封闭1小时。弃封闭液,在每孔中加入70μl噬菌体抗体库,37℃孵育2小时。弃孔中未结合噬菌体,用TBS/Tween 20溶液洗每一孔,共洗10次,可用带有吸头的移液器反复吹打每孔,但切勿过度。用蒸馏水洗两遍,将每孔中的液体吸干。每孔加入50μl甘氨酸-盐酸(pH2.2)洗脱缓冲液,室温孵育10分钟,在此期间用移液器吸头反复吹打每孔,注意切勿吹出过多气泡。加入3~5μl 2mol/lTris,使溶液的pH值在7左右,以中和洗脱下来的噬菌体溶液。立即加入2ml新鲜制备的约OD600=0.7~1.3的E.coli XL1-Blue菌,室温孵育15~20分钟。转入一200ml三角瓶中,加入含20μg/ml氨苄青霉素、10μg/ml四环素的SB培养基,立即取1μl,0.1μl,0.01μl涂LB氨苄平皿以滴定洗脱下来的噬菌体,其余部分加入100ml含100μg/ml氨苄青霉素,10μg/ml四环素的SB培养基37℃振荡培养1小时。加入1012pfu的辅助噬菌体VCSM13,37℃振荡培养2小时。加入卡那霉素70μg/ml,37℃振荡培养过夜。重复以上富集筛选步骤二轮。经二轮富集筛选后,将洗脱下来的噬菌体感染新鲜制备的感受态XL1-Blue菌,37℃过夜培养后,保存长有单个菌落的平皿于4℃待用。
抗D-二聚体单克隆抗体的制备
最后一次富集后洗脱下来的噬菌体感染新鲜制备的感受态XL1-Blue菌,37℃过夜培养后,在长有单个菌落的平皿上挑取60个菌落,接种到2ml含100μg/ml氨苄青霉素,10μg/ml四环素的SB培养基中,37℃过夜培养。次日以1:50接种于10ml含100μg/ml氨苄青霉素,10μg/ml四环素的SB培养基中,37℃培养至OD=0.1~0.5,加入辅助噬菌体,继续培养过夜。次日离心取上清,即为噬菌体抗体。将上清用4%PEG8000和3%Nacl浓缩为200μl,存放于-20℃。抗D-二聚体噬菌体抗体的筛选按购自Amersham phamacia公司的重组噬菌体抗体鉴定试剂盒说明书进行。
Fab抗体在原核细胞中的可溶性表达
经上述富集筛选后的阳性克隆,经37℃过夜培养后,提取质粒DNA并测定DNA浓度。取1μg质粒DNA,经SpeI和NheI酶切消化,电泳纯化消化后的单一条带,分子量约4700bp。重悬DNA于无菌水中,加2U连接酶及相应连接缓冲液,在总量20μl体系中,16℃连接过夜。将上述连接产物转化感受态宿主菌XL1-Blue。挑取单个菌落,培养扩增后提取质粒DNA,检测geneIII基因是否被切除:用XhoI和NotI双酶切,应切出约700bp的片段,如geneIII基因仍存在,则切出约1200bp的片段。取上述阳性菌种10μl接种入4mlSB培养液(含100μg/ml氨苄青霉素),37℃过夜培养后,转种于50mlSB培养液(含100μg/ml氨苄青霉素),37℃振荡培养至OD=0.1~0.3。加入1mmol/L IPTG,置30℃振荡培养约10~12小时。4000r/min 4℃离心15分钟。弃上清,用1/10体积PBS(pH7.4)重悬细菌沉淀,超声破碎,程序设置为:功率300W,超声4s,间歇4s,重复99次为1循环,共2~3循环。10000r/min 4℃离心20分钟。上清即含有表达的可溶性Fab抗体。
Claims (5)
1.一种产生人源抗D-二聚体的特异性导向溶栓抗体的大肠杆菌,其特征在于通过半套式PCR方法将人抗体的重链基因和轻链基因连入噬菌体质粒pComb3H形成重组噬菌体质粒pComb3H-HL,重组噬菌体质粒pComb3H-HL在辅助噬菌体VCSM13的超感染下构建成噬菌体抗体组合文库;经ELISA方法筛选出抗人纤维蛋白降解产物D-二聚体的特异株。
2.一种权利要求1所述含人源抗D-二聚体的特异性导向溶栓抗体的大肠杆菌分泌的抗体轻链的氨基酸序列,其特征在于:
S V S E S P G K T V T I S C T
G S S G S I A S N Y V Q W Y R
Q R P G S A P T T V I Y E D N
Q R P S G V P D R F S G S I D
S S T N S A S L T I S G L K T
E D E A D Y Y C Q S Y D S S N
W V F G G G T K L T V L G Q P
K A A P S V T L F P P S S E E
L Q A N K A T L V C L I S D F
Y P G A V T V A W K A D S S P
V K A G V E T T T P S K Q S N
N K Y A A S S Y L S L T P E Q
W E S H K S Y S C Q V T H E G
S T V E K T V A
3.一种产生权利要求2所述人源导向溶栓抗体的轻链的基因序列,其特征在于:
TCTGTGTCGGAGTCTCCGGGGAAGACGGTAACCATCTCCTGCACC
GGCAGCAGTGGCAGCATTGCCAGCAACTATGTGCAGTGGTACCGG
CAGCGCCCGGGCAGTGCCCCCACCACTGTGATCTATGAGGATAAC
CAAAGACCCTCTGGGGTCCCTGATCGGTTCTCTGGCTCCATCGAC
AGCTCCACCAACTCTGCCTCCCTCACCATCTCTGGACTGAAGACT
GAGGACGAGGCTGACTACTACTGTCAGTCTTATGATAGCAGCAAT
TGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCC
AAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAG
CTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTC
TACCCGGGAGCCGTGACGGTGGCCTGGAAGGCAGATAGCAGCCCC
GTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAAC
AACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAGCAG
TGGGAGTCCCACAAAAGCTACAGCTGCCAGGTCACGCATGAAGGG
AGCACCGTGGAGAAGACAGTGGCC
4.一种权利要求1所述含人源抗D-二聚体的特异性导向溶栓抗体的大肠杆菌分泌的抗体重链的氨基酸序列,其特征在于:
E S G G G V V Q P G R S L R L
S C A A S G F T F S S Y A M S
W V R Q A P G K G L E W V S A
I S G S G G S T Y Y A D S V K
G R F T I S R D N S K N T L Y
L Q M D S L R A E D T A V Y Y
C A K P H G R Y Y D S S G Y R
T Y F D Y W G Q G T L V T V S
S G S A S A P T L F P L V S C
E N S P S D T S S V A V G C L
A Q D F L P D S I T F S W K Y
K N N S D I S S T R G F P S V
L R G G K Y A A T S Q V L L P
S K D V M Q G T D E H V V C K
V Q H P N G N K E K N V P L P
V I A E L P
5.一种产生权利要求4所述人源导向溶栓抗体的重链的基因序列,其特征在于:
GAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTC
TCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGC
TGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCT
ATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAG
GGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTAT
CTGCAAATGGACAGCCTGAGAGCCGAGGACACGGCCGTATATTAC
TGTGCGAAGCCCCACGGGCGATACTATGATAGTAGTGGTTATCGA
ACCTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCC
TCAGGGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTCCTGT
GAGAATTCCCCGTCGGATACGAGCAGCGTGGCCGTTGGCTGCCTC
GCACAGGACTTCCTTCCCGACTCCATCACTTTCTCCTGGAAATAC
AAGAACAACTCTGACATCAGCAGCACCCGGGGCTTCCCATCAGTC
CTGAGAGGGGGCAAGTACGCAGCCACCTCACAGGTGCTGCTGCCT
TCCAAGGACGTCATGCAGGGCACAGACGAACACGTGGTGTGCAAA
GTCCAGCACCCCAACGGCAACAAAGAAAAGAACGTGCCTCTTCCA
GTGATTGCAGAGCTGCCT
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