法律状态公告日
法律状态信息
法律状态
2008-05-28
专利权的终止(未缴年费专利权终止)
专利权的终止(未缴年费专利权终止)
2006-11-08
授权
授权
2005-03-09
实质审查的生效
实质审查的生效
2005-01-12
公开
公开
一、技术领域
本发明属于基因工程技术领域。
二、背景技术
由于蜘蛛天然拖丝具有优于蚕丝的力学特性,除其强度能与蚕丝媲美外,最主要的优点是其同时具备蚕丝所缺乏的良好的弹性。优良的力学特性使蜘蛛拖丝在材料科学上有重要意义,尤其在某些特殊领域如医学、军事及航空航天等方面具有重要的应用价值。但由于蜘蛛独居和肉食的生活习性以及相互残杀的特点使人工大规模、高密度饲养蜘蛛以获得蛛丝的方案很难实施,而蜘蛛拖丝由大壶状腺丝蛋白分子组成,因此克隆大壶状腺丝蛋白基因进而通过基因工程方法生产重组大壶状腺丝蛋白成为首选途径。
三、发明内容
本发明需要解决的问题:
克隆悦目金蛛大壶状腺丝蛋白基因,并通过基因工程技术获得大壶状腺重组丝蛋白,为人工蜘蛛拖丝的生产提供原料。
本发明人所述悦目金蛛大壶状腺丝蛋白基因的序列是发明人在南京师范大学生命科学学院遗传资源研究所通过一系列的实验获得的。实验步骤和结果为:
1、根据其他蜘蛛大壶状腺丝蛋白的氨基酸序列设计简并引物PMaSp2:5’-GGNTANGGNCCNGGNTCNGG-3’(N=A,T,CorT),用悦目金蛛丝腺SMART RACE cDNA文库(图1)为模板,运用3′RACE技术获得了几条电泳条带(图2),每个条带分别切胶纯化后序列测定。
2、根据1获得的cDNA序列测定的结果表明,其中一条大小约为900bp的条带被证明为悦目金蛛大壶状腺丝蛋白基因的3’端功能片段(图2)。测序后获得长度为918bp的大壶状腺丝蛋白基因(Aa-MaSp2)的cDNA序列(图3),该序列由816bp的编码区(编码272个氨基酸)和99bp的3′端非翻译区组成,加尾信号AATAAA位于距离终止密码子(TAA)下游57bp处。美国国家生物技术情报中心(National Center for Biotechnology Information,NCBI)已正式接受该基因为GenBank的新成员,检索号为AY365017。
1 ggaggttatg gacccggatc tggacaacaa ggaccaggag gagccggtca acaaggacct
61 ggaggtctag gaccatatgg acctggtgca gcagcagcag ccgcaggtag ctatggacca
121 agagcaggcc aacagagacc aggagctggt caacaaggac ctggagctgg acaacaaggt
181 ccaggtggag caggccaacg aggaccgtac ggacccgggg cagcagccgc agctgcagcc
241 gctggcggct atggacaaca aggacctgga gctggacaac aaggaccagg agtaggacaa
301 caaggtccag gtggagcagg ccaacgagga ccatacggac ccggggcagc agccgcagct
361 gcagccgctg gtggctatgg accaagatat ggacaacaag gacctggagc aggaccatat
421 gggccaggag caggagccac agctgctgcc gctggaggat atggaccagg agctggacag
481 caaggaccaa gaagtcaggc tccagtcgca tccgctgcag ccgctcgact ttcttctcct
541 caagccggtt ctagagtttc atctgctgtt tcaactttgg tatcgagtgg acctactaat
601 cctgcatcac tttctaatgc tatcggtagc gttgtttcgc aggttagtgc aagcaatccc
661 ggccttccta gttgcgatgt tcttgtgcaa gcattactgg aaatcgtatc cgccctcgta
721 catatccttg gatcttccag cattggccaa attaactatt ccgcttcttc tcagtatgcc
781 cgattagttg gccaatctat tgctcaggcc cttggttaat ttcataaatg atttcaattt
841 ttctattaat gtaatgacag atttgtaatc tttctgaata aattttgaag catatttaaa
901 aaaaaaaaaa aaaaaaaaa
悦目金蛛大壶状腺丝蛋白基因cDNA序列
Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gly Ala Gly Gln Gln Gly Pro
1 6 11 16
Gly Gly Leu Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Pro
21 26 31 36
Arg Ala Gly Gln Gln Arg Pro Gly Ala Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly
41 46 51 56
Pro Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala
61 66 71 76
Ala Gly Gly Tyr Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Val Gly Gln
81 86 91 96
Gln Gly Pro Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala
101 106 111 116
Ala Ala Ala Gly Gly Tyr Gly Pro Arg Tyr Gly Gln Gln Gly Pro Gly Ala Gly Pro Tyr
121 126 131 136
Gly Pro Gly Ala Gly Ala Thr Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln
141 146 151 156
Gln Gly Pro Arg Ser Gln Ala Pro Val Ala Ser Ala Ala Ala Ala Arg Leu Ser Ser Pro
161 165 171 176
Gln Ala Gly Ser Arg Val Ser Ser Ala Val Ser Thr Leu Val Ser Ser Gly Pro Thr Asn
181 186 191 196
Pro Ala Ser Leu Ser Asn Ala Ile Gly Ser Val Val Ser Gln Val Ser Ala Ser Asn Pro
201 206 211 216
Gly Leu Pro Ser Cys Asp Val Leu Val Gln Ala Leu Leu Glu Ile Val Ser Ala Leu Val
221 226 231 236
His Ile Leu Gly Ser Ser Ser Ile Gly Gln Ile Asn Tyr Ser Ala Ser Ser Gln Tyr Ala
241 246 251 256
Arg Leu Val Gly Gln Ser Ile Ala Gln Ala Leu Gly
261 266 271
悦目金蛛大壶状腺丝蛋白氨基酸序列
3、由于蜘蛛大壶状腺丝蛋白基因大部分为重复序列,所以克隆该基因的长片段非常困难。为使通过基因工程手段获得的人工大壶状腺丝蛋白分子量尽量接近与蜘蛛自然产大壶状腺丝蛋白分子,我们利用2中得到的悦目金蛛大壶状腺丝蛋白基因cDNA序列中的功能片段设计了4个引物(primer-1:5’-GCGACGGGATCCGGAGGTTATGGACCC-3’;primer-2:5’-TAGCATGGATCCTACGACCATGGGAGGTTATGGACCC-3’;primer-3:5’-GCGATCAAGCTTGTACGAGATCTTGCTGCAGCGGCTGCAGCGGATGCGACTGGAGCC-3’;primer-4:5’-TACGTAAAGCTTTAATTTAGTGGTGGTGGTGGTGGTGACCAAGGGCCTGAGC-3’),并采用基因操作技术(图4)人工拼接了编码悦目金蛛大壶状腺丝蛋白的人工基因-I和人工基因-II,这两个编码悦目金蛛大壶状腺丝蛋白的人工基因也被美国国家生物技术情报中心(National Center for BiotechnologyInformation,NCBI)所属GenBank确认,检索号分别为AY365013和AY365014。
1 ggatcctacg accatgggag gttatggacc cggatctgga caacaaggac caggaggagc
61 cggtcaacaa ggacctggag gtctaggacc atatggacct ggtgcagcag cagcagccgc
121 aggtagctat ggaccaagag caggccaaca gagaccagga gctggtcaac aaggacctgg
181 agctggacaa caaggtccag gtggagcagg ccaacgagga ccgtacggac ccggggcagc
241 agccgcagct gcagccgctg gcggctatgg acaacaagga cctggagctg gacaacaagg
301 accaggagta ggacaacaag gtccaggtgg agcaggccaa cgaggaccat acggacccgg
361 ggcagcagcc gcagctgcag ccgctggtgg ctatggacca agatatggac aacaaggacc
421 tggagcagga ccatatgggc caggagcagg agccacagct gctgccgctg gaggatatgg
481 accaggagct ggacagcaag gaccaagaag tcaggctcca gtcgcatccg ctgcagccgc
541 tgcagcaaga tccggaggtt atggacccgg atctggacaa caaggaccag gaggagccgg
601 tcaacaagga cctggaggtc taggaccata tggacctggt gcagcagcag cagccgcagg
661 tagctatgga ccaagagcag gccaacagag accaggagct ggtcaacaag gacctggagc
721 tggacaacaa ggtccaggtg gagcaggcca acgaggaccg tacggacccg gggcagcagc
781 cgcagctgca gccgctggcg gctatggaca acaaggacct ggagctggac aacaaggacc
841 aggagtagga caacaaggtc caggtggagc aggccaacga ggaccatacg gacccggggc
901 agcagccgca gctgcagccg ctggtggcta tggaccaaga tatggacaac aaggacctgg
961 agcaggacca tatgggccag gagcaggagc cacagctgct gccgctggag gatatggacc
1021 aggagctgga cagcaaggac caagaagtca ggctccagtc gcatccgctg cagccgctcg
1081 actttcttct cctcaagccg gttctagagt ttcatctgct gtttcaactt tggtatcgag
1141 tggacctact aatcctgcat cactttctaa tgctatcggt agcgttgttt cgcaggttag
1201 tgcaagcaat cccggccttc ctagttgcga tgttcttgtg caagcattac tggaaatcgt
1261 atccgccctc gtacatatcc ttggatcttc cagcattggc caaattaact attccgcttc
1321 ttctcagtat gcccgattag ttggccaatc tattgctcag gcccttggtc accaccacca
1381 ccaccactaa gtcgacatta aagctt
编码悦目金蛛大壶状腺丝蛋白的人工基因-I DNA序列
Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gly Ala Gly Gln Gln Gly Pro
1 6 11 16
Gly Gly Leu Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Pro
21 26 31 36
Arg Ala Gly Gln Gln Arg Pro Gly Ala Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly
41 46 51 56
Pro Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala
61 66 71 76
Ala Gly Gly Tyr Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Val Gly Gln
81 86 91 96
Gln Gly Pro Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala
101 106 111 116
Ala Ala Ala Gly Gly Tyr Gly Pro Arg Tyr Gly Gln Gln Gly Pro Gly Ala Gly Pro Tyr
121 126 131 136
Gly Pro Gly Ala Gly Ala Thr Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln
141 146 151 156
Gln Gly Pro Arg Ser Gln Ala Pro Val Ala Ser Ala Ala Ala Ala Ala Ala Arg Ser Gly
161 165 171 176
Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gly Ala Gly Gln Gln Gly Pro Gly
181 186 191 196
Gly Leu Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Pro Arg
201 206 211 216
Ala Gly Gln Gln Arg Pro Gly Ala Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly Pro
221 226 231 236
Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala Ala
241 246 251 256
Gly Gly Tyr Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Val Gly Gln Gln
261 266 271 276
Gly Pro Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala
281 286 291 296
Ala Ala Gly Gly Tyr Gly Pro Arg Tyr Gly Gln Gln Gly Pro Gly Ala Gly Pro Tyr Gly
301 306 311 316
Pro Gly Ala Gly Ala Thr Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln
321 326 331 336
Gly Pro Arg Ser Gln Ala Pro Val Ala Ser Ala Ala Ala Ala Arg Leu Ser Ser Pro Gln
341 346 351 356
Ala Gly Ser Arg Val Ser Ser Ala Val Ser Thr Leu Val Ser Ser Gly Pro Thr Asn Pro
361 366 371 376
Ala Ser Leu Ser Asn Ala Ile Gly Ser Val Val Ser Gln Val Ser Ala Ser Asn Pro Gly
381 386 391 396
Leu Pro Ser Cys Asp Val Leu Val Gln Ala Leu Leu Glu Ile Val Ser Ala Leu Val His
401 406 411 416
Ile Leu Gly Ser Ser Ser Ile Gly Gln Ile Asn Tyr Ser Ala Ser Ser Gln Tyr Ala Arg
421 426 431 436
Leu Val Gly Gln Ser Ile Ala Gln Ala Leu Gly His His His His His His
441 446 451 456
人工基因-I编码的悦目金蛛大壶状腺丝蛋白氨基酸序列
1 ggatcctacg accatgggag gttatggacc cggatctgga caacaaggac caggaggagc
61 cggtcaacaa ggacctggag gtctaggacc atatggacct ggtgcagcag cagcagccgc
121 aggtagctat ggaccaagag caggccaaca gagaccagga gctggtcaac aaggacctgg
181 agctggacaa caaggtccag gtggagcagg ccaacgagga ccgtacggac ccggggcagc
241 agccgcagct gcagccgctg gcggctatgg acaacaagga cctggagctg gacaacaagg
301 accaggagta ggacaacaag gtccaggtgg agcaggccaa cgaggaccat acggacccgg
361 ggcagcagcc gcagctgcag ccgctggtgg ctatggacca agatatggac aacaaggacc
421 tggagcagga ccatatgggc caggagcagg agccacagct gctgccgctg gaggatatgg
481 accaggagct ggacagcaag gaccaagaag tcaggctcca gtcgcatccg ctgcagccgc
541 tgcagcaaga tccggaggtt atggacccgg atctggacaa caaggaccag gaggagccgg
601 tcaacaagga cctggaggtc taggaccata tggacctggt gcagcagcag cagccgcagg
661 tagctatgga ccaagagcag gccaacagag accaggagct ggtcaacaag gacctggagc
721 tggacaacaa ggtccaggtg gagcaggcca acgaggaccg tacggacccg gggcagcagc
781 cgcagctgca gccgctggcg gctatggaca acaaggacct ggagctggac aacaaggacc
841 aggagtagga caacaaggtc caggtggagc aggccaacga ggaccatacg gacccggggc
901 agcagccgca gctgcagccg ctggtggcta tggaccaaga tatggacaac aaggacctgg
961 agcaggacca tatgggccag gagcaggagc cacagctgct gccgctggag gatatggacc
1021 aggagctgga cagcaaggac caagaagtca ggctccagtc gcatccgctg cagccgctgc
1081 agcaagatcc ggaggttatg gacccggatc tggacaacaa ggaccaggag gagccggtca
1141 acaaggacct ggaggtctag gaccatatgg acctggtgca gcagcagcag ccgcaggtag
1201 ctatggacca agagcaggcc aacagagacc aggagctggt caacaaggac ctggagctgg
1261 acaacaaggt ccaggtggag caggccaacg aggaccgtac ggacccgggg cagcagccgc
1321 agctgcagcc gctggcggct atggacaaca aggacctgga gctggacaac aaggaccagg
1381 agtaggacaa caaggtccag gtggagcagg ccaacgagga ccatacggac ccggggcagc
1441 agccgcagct gcagccgctg gtggctatgg accaagatat ggacaacaag gacctggagc
1501 aggaccatat gggccaggag caggagccac agctgctgcc gctggaggat atggaccagg
1561 agctggacag caaggaccaa gaagtcaggc tccagtcgca tccgctgcag ccgctcgact
1621 ttcttctcct caagccggtt ctagagtttc atctgctgtt tcaactttgg tatcgagtgg
1681 acctactaat cctgcatcac tttctaatgc tatcggtagc gttgtttcgc aggttagtgc
1741 aagcaatccc ggccttccta gttgcgatgt tcttgtgcaa gcattactgg aaatcgtatc
1801 cgccctcgta catatccttg gatcttccag cattggccaa attaactatt ccgcttcttc
1861 tcagtatgcc cgattagttg gccaatctat tgctcaggcc cttggtcacc accaccacca
1921 ccactaagtc gacattaaag ctt
编码悦目金蛛大壶状腺丝蛋白的人工基因-II DNA序列
Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gly Ala Gly Gln Gln Gly Pro
1 6 11 16
Gly Gly Leu Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Pro
21 26 31 36
Arg Ala Gly Gln Gln Arg Pro Gly Ala Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly
41 46 51 56
Pro Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala
61 66 71 76
Ala Gly Gly Tyr Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Val Gly Gln
81 86 91 96
Gln Gly Pro Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala
101 106 111 116
Ala Ala Ala Gly Gly Tyr Gly Pro Arg Tyr Gly Gln Gln Gly Pro Gly Ala Gly Pro Tyr
121 126 131 136
Gly Pro Gly Ala Gly Ala Thr Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln
141 146 151 156
Gln Gly Pro Arg Ser Gln Ala Pro Val Ala Ser Ala Ala Ala Ala Ala Ala Arg Ser Gly
161 165 171 176
Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gly Ala Gly Gln Gln Gly Pro Gly
181 186 191 196
Gly Leu Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Pro Arg
201 206 211 216
Ala Gly Gln Gln Arg Pro Gly Ala Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly Pro
221 226 231 236
Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala Ala
241 246 251 256
Gly Gly Tyr Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Val Gly Gln Gln
261 266 271 276
Gly Pro Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala
281 286 291 296
Ala Ala Gly Gly Tyr Gly Pro Arg Tyr Gly Gln Gln Gly Pro Gly Ala Gly Pro Tyr Gly
301 306 311 316
Pro Gly Ala Gly Ala Thr Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln
321 326 331 336
Gly Pro Arg Ser Gln Ala Pro Val Ala Ser Ala Ala Ala Ala Ala Ala Arg Ser Gly Gly
341 346 351 356
Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gly Ala Gly Gln Gln Gly Pro Gly Gly
361 366 371 376
Leu Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Pro Arg Ala
381 386 391 396
Gly Gln Gln Arg Pro Gly Ala Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly
401 406 411 416
Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala Ala Gly
421 426 431 436
Gly Tyr Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Val Gly Gln Gln Gly
441 446 451 456
Pro Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala
461 466 471 476
Ala Gly Gly Tyr Gly Pro Arg Tyr Gly Gln Gln Gly Pro Gly Ala Gly Pro Tyr Gly Pro
481 486 491 496
Gly Ala Gly Ala Thr Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly
501 506 511 516
Pro Arg Ser Gln Ala Pro Val Ala Ser Ala Ala Ala Ala Arg Leu Ser Ser Pro Gln Ala
521 526 531 536
Gly Ser Arg Val Ser Ser Ala Val Ser Thr Leu Val Ser Ser Gly Pro Thr Asn Pro Ala
541 546 551 556
Ser Leu Ser Asn Ala Ile Gly Ser Val Val Ser Gln Val Ser Ala Ser Asn Pro Gly Leu
561 566 571 576
Pro Ser Cys Asp Val Leu Val Gln Ala Leu Leu Glu Ile Val Ser Ala Leu Val His Ile
581 585 591 596
Leu Gly Ser Ser Ser Ile Gly Gln Ile Asn Tyr Ser Ala Ser Ser Gln Tyr Ala Arg Leu
601 606 611 616
Val Gly Gln Ser Ile Ala Gln Ala Leu Gly His His His His His His
621 626 631 636
人工基因-II编码的悦目金蛛大壶状腺丝蛋白氨基酸序列
本发明技术方案:
l、悦目金蛛大壶状腺丝蛋白的制备
(1)以提取的悦目金蛛丝腺总RNA为模板,用SMARTTM cDNA Library Construction Kit试剂盒(购自Clontech公司)反转录合成悦目金蛛丝腺SMART RACE cDNA文库。利用简便引物PMaSp2:5’-GGNTANGGNCCNGGNTCNGG-3’(N=A,T,CorT)和试剂盒提供的接头引物3’PCRPrimer(5’-TTCTAGAGGCCGAGGCGGCC-3’)进行3’RACE,把PCR产物切胶纯化后连接于pMDl8-T载体(购自Takara公司),通过序列测定鉴定后获得含有悦目金蛛大壶状腺丝蛋白基因的重组质粒pMD18-T-MaSp2。
(2)用引物5’-GCACGACCATGGGAGGTTATGGACCC-3’和5’-TAATGGATCCTTAGTGGTGGTGGTGGTGGTGACCAAGGGCCTGAGCA-3’以上述重组质粒pMDl8-T-MaSp2为模板扩增出悦目金蛛大壶状腺丝蛋白基因的编码区。反应条件:95℃预变性5min,95℃变性30s,60℃退火30s,72℃延伸1min,30个循环,72℃延伸10min。
(3)上述扩增产物割胶纯化后与表达载体pET28 vector(购自Novagen公司)同时用NcoI和BamHI在37℃双酶切数小时。琼脂糖凝胶电泳回收酶切片段及pET28载体后用T4DNA连接酶连接。将连接产物转化大肠杆菌DH5α,通过双酶切鉴定重组表达质粒pET28-MaSp2(图5)。
(4)将重组质粒pET28-MaSp2转化大肠杆菌BL21(DE3)菌株后,以IPTG诱导若干小时,离心后加上样缓冲液煮沸破碎细胞,进行12%浓度的SDS-PAGE电泳(图6)。
(5)利用Ni-NTA His-bind亲和层析柱(购自Novagen公司)对重组蛋白进行初步分离纯化,然后用分子筛技术进行二次纯化。
2、悦目金蛛大壶状腺丝蛋白多克隆抗体的制备
将重组蛋白与佐剂(Complete/Incomplete Freund’s Adjuvant)等量混合,免疫新西兰兔(背部皮下注射),每隔3-4周激发注射一次,共三次,于3个月后取血分离血清。第一次免疫使用完全佐剂,后几次免疫使用不完全佐剂。以免疫前动物血清作为对照做ELISA,测定各免疫兔血清中抗体滴度。筛选高滴度血清,进一步用抗原作竞争抑制免疫试验确定抗体的特异性。
3、编码大壶状腺丝蛋白的人工基因-I和人工基因-II的表达
(1)在通过基因操作技术拼接编码大壶状腺丝蛋白的人工基因-I和人工基因-II的过程中(图4),这两个人工基因已被设计与表达质粒pET28结合,形成的重组表达质粒分别为pET28-MaSp2(2+1)和pET28-MaSp2(3+1)。
(2)把重组表达质粒pET28-MaSp2(2+1)和pET28-MaSp2(3+1)分别转化大肠杆菌BL21(DE3)菌株后,以IPTG诱导若干小时,离心后加上样缓冲液煮沸破碎细胞,进行12%浓度的SDS-PAGE电泳(图7,8)。
(3)利用Ni-NTA His-bind亲和层析柱(购自Novagen公司)对重组蛋白进行初步分离纯化,然后用分子筛技术进行二次纯化。
4、重组大壶状腺丝蛋白的鉴定
(1)把纯化后的三种重组丝蛋白MaSp2,MaSp2(2+1)和MaSp2(3+1)分别于110℃下用浓盐酸水解24小时,再在日立835-50型氨基酸分析仪上分析它们的氨基酸组成和含量。
(2)把纯化后的三种重组丝蛋白MaSp2,MaSp2(2+1)和MaSp2(3+1)分别进行12%浓度的SDS-PAGE电泳,再通过蛋白质转移的方法转至PVDF膜上,在ProciseTM491型蛋白质序列测定仪(购自PE/ABI公司)上分别测定每种重组丝蛋白的N端10个氨基酸残基的序列。
(3)纯化后的三种重组丝蛋白MaSp2,MaSp2(2+1)和MaSp2(3+1)分别进行12%浓度的SDS-PAGE电泳,再通过蛋白质转移的方法转至PVDF膜上,利用悦目金蛛大壶状腺丝蛋白多克隆抗体进行Western分析以鉴定重组丝蛋白(图9)。
5、保持重组大壶状腺丝蛋白稳定溶解性的缓冲液体系的筛选及人工拖丝的研制
由于大壶状腺丝蛋白分子有许多连续的Ala残基组成的重复片断,从而具有自发形成不溶于水的β-折叠二级结构的倾向。但根据蜘蛛自然蛛丝形成机理,丝蛋白在从纺孔排出体外、转变为固态丝之前始终处于浓蛋白水溶液的状态,所以筛选合适的、能高浓度溶解重组丝蛋白的缓冲液体系非常必要。用饱和硫酸氨沉淀重组丝蛋白后用6M盐酸胍溶解丝蛋白沉淀,再用不同参数的缓冲液进行透析以观察丝蛋白在各种缓冲液中的溶解性。通过这种方法已经筛选到PH值为2-4的一种缓冲液体系(50mmol/L NaCl;3.0mmol/L KCl:l0mmol/L Na2HPO4;3mmol/KH2PO4)能溶解20-30%的重组丝蛋白,从而解决了人工拖丝研制中保持重组丝蛋白在水中溶解性这个关键问题。人工拖丝可开发成为一种在材料科学、医学、军事及航空航天等领域有应用价值的可降解的新型生物材料。
四、附图说明
图1、悦目金蛛丝腺SMART RACE cDNA文库电泳图(M:DNA标记;1:双链cDNA)
图2、3’RACE PCR结果(M:DNA标记;l:3’RACE PCR产物;箭头所指条带为目的条带)
图3、悦目金蛛大壶状腺丝蛋白基因cDNA序列及编码蛋白的氨基酸序列
图4、编码大壶状腺丝蛋白的人工基因-I和人工基因-II的拼接过程示意图
图5、重组表达质粒的限制性酶切分析(M:DNA分子量标记;1:pET28-MaSp2;2:pET28-MaSp2经NcoI和BamHI双酶切;3:pET28-MaSp2(2+1);4:pET28-MaSp2(2+1)经NcoI和HindIII双酶切;5:pET28-MaSp2(3+1);6:pET28-MaSp2(3+1)经NcoI和HindIII双酶切)
图6、悦目金蛛大壶状腺丝蛋白诱导表达及蛋白质纯化的聚丙烯酰氨凝胶电泳分析(1:BL21(DE3)+pET28;2:BL21(DE3)+pET28-MaSp2经IPTG诱导0小时;3:BL21(DE3)+pET28-MaSp2经IPTG诱导4小时;4:蛋白质分子量标记;5:BL21(DE3)+pET28-MaSp2经IPTG诱导4小时后细菌破碎后的上清液;6:BL21(DE3)+pET28-MaSp2经IPTG诱导4小时后细菌破碎后的沉淀;7:重组蛋白经Ni-NTA His-bind亲和层析柱初步纯化;8:重组蛋白经分子筛技术进行二次纯化)
图7、人工基因-I诱导表达及蛋白质纯化的聚丙烯酰氨凝胶电泳分析(1:BL21(DE3)+pET28;2:BL21(DE3)+pET28-MaSp2(2+1)经IPTG诱导0小时;3:BL21(DE3)+pET28-MaSp2(2+1)经IPTG诱导4小时;4:蛋白质分子量标记;5:BL21(DE3)+pET28-MaSp2(2+1)经IPTG诱导4小时后细菌破碎后的上清液;6:BL21(DE3)+pET28-MaSp2(2+1)经IPTG诱导4小时后细菌破碎后的沉淀;7:重组蛋白经Ni-NTA His-bind亲和层析柱初步纯化;8:重组蛋白经分子筛技术进行二次纯化)
图8、人工基因-II诱导表达及蛋白质纯化的聚丙烯酰氨凝胶电泳分析(1:BL21(DE3)+pET28;2:BL21(DE3)+pET28-MaSp2(3+1)经IPTG诱导0小时;3:BL21(DE3)+pET28-MaSp2(3+1)经IPTG诱导4小时;4:蛋白质分子量标记;5:BL21(DE3)+pET28-MaSp2(3+1)经IPTG诱导4小时后细菌破碎后的上清液;6:BL21(DE3)+pET28-MaSp2(3+1)经IPTG诱导4小时后细菌破碎后的沉淀;7:重组蛋白经Ni-NTA His-bind亲和层析柱初步纯化;8:重组蛋白经分子筛技术进行二次纯化)
图9、重组大壶状腺丝蛋白的Western分析(1:蛋白质分子量标记;2:MaSp2;3:MaSp2(2+1);4:MaSp2(3+1);5:阴性对照)
悦目金蛛大壶状腺丝蛋白基因序列表
<110>南京师范大学
<120>悦目金蛛大壶状腺丝蛋白基因(Aa-MaSp2)
<160>3
<210>1
<211>918bp
<212>cDNA
<213>悦目金蛛(Argiope amoena)
<400>1
0 ggaggttatg gacccggatc tggacaacaa ggaccaggag gagccggtca acaaggacct
61 ggaggtctag gaccatatgg acctggtgca gcagcagcag ccgcaggtag ctatggacca
121 agagcaggcc aacagagacc aggagctggt caacaaggac ctggagctgg acaacaaggt
181 ccaggtggag caggccaacg aggaccgtac ggacccgggg cagcagccgc agctgcagcc
241 gctggcggct atggacaaca aggacctgga gctggacaac aaggaccagg agtaggacaa
301 caaggtccag gtggagcagg ccaacgagga ccatacggac ccggggcagc agccgcagct
361 gcagccgctg gtggctatgg accaagatat ggacaacaag gacctggagc aggaccatat
421 gggccaggag caggagccac agctgctgcc gctggaggat atggaccagg agctggacag
481 caaggaccaa gaagtcaggc tccagtcgca tccgctgcag ccgctcgact ttcttctcct
541 caagccggtt ctagagtttc atctgctgtt tcaactttgg tatcgagtgg acctactaat
601 cctgcatcac tttctaatgc tatcggtagc gttgtttcgc aggttagtgc aagcaatccc
661 ggccttccta gttgcgatgt tcttgtgcaa gcattactgg aaatcgtatc cgccctcgta
721 catatccttg gatcttccag cattggccaa attaactatt ccgcttcttc tcagtatgcc
781 cgattagttg gccaatctat tgctcaggcc cttggttaat ttcataaatg atttcaattt
841 ttctattaat gtaatgacag atttgtaatc tttctgaata aattttgaag catatttaaa
901 aaaaaaaaaa aaaaaaaaa
悦目金蛛大壶状腺丝蛋白基因cDNA序列
Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gly Ala Gly Gln Gln Gly Pro
1 6 11 16
Gly Gly Leu Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Pro
21 26 31 36
Arg Ala Gly Gln Gln Arg Pro Gly Ala Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly
41 46 51 56
Pro Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala
61 66 71 76
Ala Gly Gly Tyr Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Val Gly Gln
81 86 91 96
Gln Gly Pro Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala
101 106 111 116
Ala Ala Ala Gly Gly Tyr Gly Pro Arg Tyr Gly Gln Gln Gly Pro Gly Ala Gly Pro Tyr
121 126 131 136
Gly Pro Gly Ala Gly Ala Thr Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln
141 146 151 156
Gln Gly Pro Arg Ser Gln Ala Pro Val Ala Ser Ala Ala Ala Ala Arg Leu Ser Ser Pro
161 165 171 176
Gln Ala Gly Ser Arg Val Ser Ser Ala Val Ser Thr Leu Val Ser Ser Gly Pro Thr Asn
181 186 191 196
Pro Ala Ser Leu Ser Asn Ala Ile Gly Ser Val Val Ser Gln Val Ser Ala Ser Asn Pro
201 206 211 216
Gly Leu Pro Ser Cys Asp Val Leu Val Gln Ala Leu Leu Glu Ile Val Ser Ala Leu Val
221 226 231 236
His Ile Leu Gly Ser Ser Ser Ile Gly Gln Ile Asn Tyr Ser Ala Ser Ser Gln Tyr Ala
241 246 251 256
Arg Leu Val Gly Gln Ser Ile Ala Gln Ala Leu Gly
261 266 271
悦目金蛛大壶状腺丝蛋白氨基酸序列
编码悦目金蛛大壶状腺丝蛋白的人工基因I序列表
<110>南京师范大学
<120>编码悦目金蛛大壶状腺丝蛋白的人工基因I
<160>3
<210>2
<21l>1406bp
<212>DNA
<213>人工序列
<400>1
0 ggatcctacg accatgggag gttatggacc cggatctgga caacaaggac caggaggagc
61 cggtcaacaa ggacctggag gtctaggacc atatggacct ggtgcagcag cagcagccgc
121 aggtagctat ggaccaagag caggccaaca gagaccagga gctggtcaac aaggacctgg
181 agctggacaa caaggtccag gtggagcagg ccaacgagga ccgtacggac ccggggcagc
241 agccgcagct gcagccgctg gcggctatgg acaacaagga cctggagctg gacaacaagg
301 accaggagta ggacaacaag gtccaggtgg agcaggccaa cgaggaccat acggacccgg
361 ggcagcagcc gcagctgcag ccgctggtgg ctatggacca agatatggac aacaaggacc
421 tggagcagga ccatatgggc caggagcagg agccacagct gctgccgctg gaggatatgg
481 accaggagct ggacagcaag gaccaagaag tcaggctcca gtcgcatccg ctgcagccgc
541 tgcagcaaga tccggaggtt atggacccgg atctggacaa caaggaccag gaggagccgg
601 tcaacaagga cctggaggtc taggaccata tggacctggt gcagcagcag cagccgcagg
661 tagctatgga ccaagagcag gccaacagag accaggagct ggtcaacaag gacctggagc
721 tggacaacaa ggtccaggtg gagcaggcca acgaggaccg tacggacccg gggcagcagc
781 cgcagctgca gccgctggcg gctatggaca acaaggacct ggagctggac aacaaggacc
841 aggagtagga caacaaggtc caggtggagc aggccaacga ggaccatacg gacccggggc
901 agcagccgca gctgcagccg ctggtggcta tggaccaaga tatggacaac aaggacctgg
961 agcaggacca tatgggccag gagcaggagc cacagctgct gccgctggag gatatggacc
1021 aggagctgga cagcaaggac caagaagtca ggctccagtc gcatccgctg cagccgctcg
1081 actttcttct cctcaagccg gttctagagt ttcatctgct gtttcaactt tggtatcgag
1141 tggacctact aatcctgcat cactttctaa tgctatcggt agcgttgttt cgcaggttag
1201 tgcaagcaat cccggccttc ctagttgcga tgttcttgtg caagcattac tggaaatcgt
1261 atccgccctc gtacatatcc ttggatcttc cagcattggc caaattaact attccgcttc
1321 ttctcagtat gcccgattag ttggccaatc tattgctcag gcccttggtc accaccacca
1381 ccaccactaa gtcgacatta aagctt
编码悦目金蛛大壶状腺丝蛋白的人工基因-I DNA序列
Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gly Ala Gly Gln Gln Gly Pro
l 6 11 16
Gly Gly Leu Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Pro
21 26 31 36
Arg Ala Gly Gln Gln Arg Pro Gly Ala Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly
41 46 51 56
Pro Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala
61 66 71 76
Ala Gly Gly Tyr Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Val Gly Gln
81 86 91 96
Gln Gly Pro Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala
101 106 111 116
Ala Ala Ala Gly Gly Tyr Gly Pro Arg Tyr Gly Gln Gln Gly Pro Gly Ala Gly Pro Tyr
121 126 131 136
Gly Pro Gly Ala Gly Ala Thr Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln
141 146 151 156
Gln Gly Pro Arg Ser Gln Ala Pro Val Ala Ser Ala Ala Ala Ala Ala Ala Arg Ser Gly
161 165 171 176
Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gly Ala Gly Gln Gln Gly Pro Gly
181 186 191 196
Gly Leu Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Pro Arg
201 206 211 216
Ala Gly Gln Gln Arg Pro Gly Ala Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly Pro
221 226 231 236
Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala Ala
241 246 251 256
Gly Gly Tyr Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Val Gly Gln Gln
261 266 271 276
Gly Pro Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala
281 286 291 296
Ala Ala Gly Gly Tyr Gly Pro Arg Tyr Gly Gln Gln Gly Pro Gly Ala Gly Pro Tyr Gly
301 306 311 316
Pro Gly Ala Gly Ala Thr Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln
321 326 331 336
Gly Pro Arg Ser Gln Ala Pro Val Ala Ser Ala Ala Ala Ala Arg Leu Ser Ser Pro Gln
341 346 351 356
Ala Gly Ser Arg Val Ser Ser Ala Val Ser Thr Leu Val Ser Ser Gly Pro Thr Asn Pro
361 366 371 376
Ala Ser Leu Ser Asn Ala Ile Gly Ser Val Val Ser Gln Val Ser Ala Ser Asn Pro Gly
381 386 391 396
Leu Pro Ser Cys Asp Val Leu Val Gln Ala Leu Leu Glu Ile Val Ser Ala Leu Val His
401 406 411 416
Ile Leu Gly Ser Ser Ser Ile Gly Gln Ile Asn Tyr Ser Ala Ser Ser Gln Tyr Ala Arg
421 426 431 436
Leu Val Gly Gln Ser Ile Ala Gln Ala Leu Gly His His His His His His
441 446 451 456
人工基因-I编码的悦目金蛛大壶状腺丝蛋白氨基酸序列
编码悦目金蛛大壶状腺丝蛋白的人工基因II序列表
<110>南京师范大学
<120>编码悦目金蛛大壶状腺丝蛋白的人工基因II
<160>3
<210>3
<211>1943bp
<212>DNA
<213>人工序列
<400>1
1 ggatcctacg accatgggag gttatggacc cggatctgga caacaaggac caggaggagc
61 cggtcaacaa ggacctggag gtctaggacc atatggacct ggtgcagcag cagcagccgc
121 aggtagctat ggaccaagag caggccaaca gagaccagga gctggtcaac aaggacctgg
181 agctggacaa caaggtccag gtggagcagg ccaacgagga ccgtacggac ccggggcagc
241 agccgcagct gcagccgctg gcggctatgg acaacaagga cctggagctggacaacaagg
301 accaggagta ggacaacaag gtccaggtgg agcaggccaa cgaggaccat acggacccgg
361 ggcagcagcc gcagctgcag ccgctggtgg ctatggacca agatatggac aacaaggacc
421 tggagcagga ccatatgggc caggagcagg agccacagct gctgccgctg gaggatatgg
481 accaggagct ggacagcaag gaccaagaag tcaggctcca gtcgcatccg ctgcagccgc
541 tgcagcaaga tccggaggtt atggacccgg atctggacaa caaggaccag gaggagccgg
601 tcaacaagga cctggaggtc taggaccata tggacctggt gcagcagcag cagccgcagg
661 tagctatgga ccaagagcag gccaacagag accaggagct ggtcaacaag gacctggagc
721 tggacaacaa ggtccaggtg gagcaggcca acgaggaccg tacggacccg gggcagcagc
781 cgcagctgca gccgctggcg gctatggaca acaaggacct ggagctggac aacaaggacc
841 aggagtagga caacaaggtc caggtggagc aggccaacga ggaccatacg gacccggggc
901 agcagccgca gctgcagccg ctggtggcta tggaccaaga tatggacaac aaggacctgg
961 agcaggacca tatgggccag gagcaggagc cacagctgct gccgctggag gatatggacc
1021 aggagctgga cagcaaggac caagaagtca ggctccagtc gcatccgctg cagccgctgc
1081 agcaagatcc ggaggttatg gacccggatc tggacaacaa ggaccaggag gagccggtca
1141 acaaggacct ggaggtctag gaccatatgg acctggtgca gcagcagcag ccgcaggtag
1201 ctatggacca agagcaggcc aacagagacc aggagctggt caacaaggac ctggagctgg
1261 acaacaaggt ccaggtggag caggccaacg aggaccgtac ggacccgggg cagcagccgc
1321 agctgcagcc gctggcggct atggacaaca aggacctgga gctggacaac aaggaccagg
1381 agtaggacaa caaggtccag gtggagcagg ccaacgagga ccatacggac ccggggcagc
1441 agccgcagct gcagccgctg gtggctatgg accaagatat ggacaacaag gacctggagc
1501 aggaccatat gggccaggag caggagccac agctgctgcc gctggaggat atggaccagg
1561 agctggacag caaggaccaa gaagtcaggc tccagtcgca tccgctgcag ccgctcgact
1621 ttcttctcct caagccggtt ctagagtttc atctgctgtt tcaactttgg tatcgagtgg
1681 acctactaat cctgcatcac tttctaatgc tatcggtagc gttgtttcgc aggttagtgc
1741 aagcaatccc ggccttccta gttgcgatgt tcttgtgcaagcattactggaaatcgtatc
1801 cgccctcgta catatccttg gatcttccag cattggccaa attaactatt ccgcttcttc
1861 tcagtatgcc cgattagttg gccaatctat tgctcaggcc cttggtcacc accaccacca
1921 ccactaagtc gacattaaag ctt
编码悦目金蛛大壶状腺丝蛋白的人工基因-II DNA序列
Gly Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gly Ala Gly Gln Gln Gly Pro
1 6 11 16
Gly Gly Leu Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Pro
21 26 31 36
Arg Ala Gly Gln Gln Arg Pro Gly Ala Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly
41 46 51 56
Pro Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala
61 66 71 76
Ala Gly Gly Tyr Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Val Gly Gln
81 86 91 96
Gln Gly Pro Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala
101 106 111 116
Ala Ala Ala Gly Gly Tyr Gly Pro Arg Tyr Gly Gln Gln Gly Pro Gly Ala Gly Pro Tyr
121 126 131 136
Gly Pro Gly Ala Gly Ala Thr Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln
141 146 151 156
Gln Gly Pro Arg Ser Gln Ala Pro Val Ala Ser Ala Ala Ala Ala Ala Ala Arg Ser Gly
161 165 171 176
Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gly Ala Gly Gln Gln Gly Pro Gly
181 186 191 196
Gly Leu Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Pro Arg
201 206 211 216
Ala Gly Gln Gln Arg Pro Gly Ala Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly Pro
221 226 231 236
Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala Ala
241 246 251 256
Gly Gly Tyr Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Val Gly Gln Gln
261 266 271 276
Gly Pro Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala
281 286 291 296
Ala Ala Gly Gly Tyr Gly Pro Arg Tyr Gly Gln Gln Gly Pro Gly Ala Gly Pro Tyr Gly
301 306 311 316
Pro Gly Ala Gly Ala Thr Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln
321 326 331 336
Gly Pro Arg Ser Gln Ala Pro Val Ala Ser Ala Ala Ala Ala Ala Ala Arg Ser Gly Gly
341 346 351 356
Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gly Ala Gly Gln Gln Gly Pro Gly Gly
361 366 371 376
Leu Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Gly Ser Tyr Gly Pro Arg Ala
381 386 391 396
Gly Gln Gln Arg Pro Gly Ala Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly
401 406 411 416
Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala Ala Gly
421 426 431 436
Gly Tyr Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Val Gly Gln Gln Gly
441 446 451 456
Pro Gly Gly Ala Gly Gln Arg Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala
461 466 471 476
Ala Gly Gly Tyr Gly Pro Arg Tyr Gly Gln Gln Gly Pro Gly Ala Gly Pro Tyr Gly Pro
481 486 491 496
Gly Ala Gly Ala Thr Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly
501 506 511 516
Pro Arg Ser Gln Ala Pro Val Ala Ser Ala Ala Ala Ala Arg Leu Ser Ser Pro Gln Ala
521 526 531 536
Gly Ser Arg Val Ser Ser Ala Val Ser Thr Leu Val Ser Ser Gly Pro Thr Asn Pro Ala
541 546 551 556
Ser Leu Ser Asn Ala Ile Gly Ser Val Val Ser Gln Val Ser Ala Ser Asn Pro Gly Leu
561 566 571 576
Pro Ser Cys Asp Val Leu Val Gln Ala Leu Leu Glu Ile Val Ser Ala Leu Val His Ile
581 585 591 596
Leu Gly Ser Ser Ser Ile Gly Gln Ile Asn Tyr Ser Ala Ser Ser Gln Tyr Ala Arg Leu
601 606 611 616
Val Gly Gln Ser Ile Ala Gln Ala Leu Gly His His His His His His
621 626 631 636
人工基因-II编码的悦目金蛛大壶状腺丝蛋白氨基酸序列
机译: 前列腺的蛋白抑制剂肿瘤和分离纯化的dna顺序编码重组载体及其表达载体,系统宿主载体产生多肽或蛋白质,药物制剂,dna探针,分离纯化的蛋白及其制备方法,抗体和方法检测的无ptsG蛋白在肿瘤细胞,rna探针外部
机译: 包含前列腺肿瘤抑制蛋白和重组载体及包含其的表达载体的分离纯化的DNA序列,用于生产多肽或蛋白的系统载体,药物制备,DNA探针,产物及其制备方法检测体外肿瘤细胞中PTSG蛋白缺失的方法和RNA探针
机译: 重组质粒pER-TB4GyrA-AcSer编码丝氨酸乙酰转移酶,可在体内乙酰化N-末端丝氨酰-胸腺嘧啶β4和杂交蛋白,可自催化分解为人胸腺蛋白酶30 /大肠杆菌E30 /β4,形成人胸腺蛋白酶30。蛋白质和基因工程人胸腺肽的制备方法