CN114349836B - 一种粘附力强的类贻贝粘蛋白及其制备方法 - Google Patents
一种粘附力强的类贻贝粘蛋白及其制备方法 Download PDFInfo
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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Abstract
本发明公开了一种粘附力强的类贻贝粘蛋白及其制备方法，属于粘附蛋白领域。本发明提供的类贻贝粘蛋白中间体mlp353，mlp316及mlp319的pI均在10左右，且含有高达23.1％～25.5％摩尔百分数的酪氨酸(Tyr)。本发明将上述类贻贝粘蛋白中间体经酪氨酸酶氧化后得到了新的类贻贝粘蛋白，与天然贻贝粘蛋白相比，本发明的类贻贝粘蛋白具有高粘附力和均匀的分子量分布，可以用来制备高粘附力的粘附材料，在海洋工程、生物医学及表面化学等领域具有广阔的应用前景。
Description
技术领域
本发明属于粘附蛋白领域，具体涉及一种粘附力强的类贻贝粘蛋白及其制备方法。
背景技术
贻贝是一种广泛分布于沿海和近海的甲壳类海洋生物，其足丝腺能分泌足丝，并通过足丝形成的足丝盘将贻贝固定在各种基材表面，研究表明，这与贻贝分泌的足丝有关。其足丝的主要成分为贻贝足丝蛋白(Mytilus edulis foot protein，Mfp)，也称为贻贝粘蛋白(mussel adhesive protein，MAP)，该蛋白具有高强度、高韧性和防水性。贻贝粘蛋白所形成的涂层具有多功能性，对多级别材料均能实现表面改性。贻贝粘蛋白还具有耐腐蚀性强、生物相容性良好、不会产生免疫反应等特点，在海洋工程、生物医学及表面化学等领域具有广泛的应用前景。
目前虽然还没有完全弄清楚贻贝粘蛋白的粘附机理，但大量的研究表明贻贝粘蛋白能粘附于几乎所有的固体材料表面，关键在于其结构中所富含的3，4-二羟基苯丙氨酸(DOPA，多巴)功能元。DOPA是一种侧基为儿茶酚(邻苯二酚)官能团的氨基酸，DOPA的苯酚基团具有很强的金属螯合能力，能在材料表面形成不可逆的有机金属络合物，还能与蛋白质等极性聚合物形成很强的氢键。因此，DOPA对贻贝粘蛋白的防水性黏合及内聚力起关键作用。
天然贻贝粘蛋白均来自于贻贝足丝分泌的一系列含有多巴基团的粘性蛋白，这些蛋白的DOPA含量从2％到28％不等，分子量的分布也从5-7KDa至108KDa不等，因此天然提取获得的贻贝粘蛋白的粘附性较低，而且具有不均一性，导致天然贻贝粘蛋白在实际应用中受限。为了克服天然贻贝粘蛋白的不足，研究者们开始研究人工合成的类贻贝粘蛋白。D.S.Hwang等(Biomaterials 28(2007)3560-3568)公开了一种类贻贝粘蛋白fp-151(即下文所述的类贻贝粘蛋白MLAP151)，它的生物相容性和溶解性优良，同时具有比天然贻贝粘蛋白更强的粘附能力。但是，该类贻贝粘蛋白fp-151的粘附能力还无法满足需求，有待进一步提高。
开发出具有更高粘附力的类贻贝粘蛋白具有重要意义。
发明内容
本发明的目的在于提供一种粘附力强的类贻贝粘蛋白及其制备方法。
本发明提供了一种基因，其核苷酸序列如SEQ ID NO.4、SEQ ID NO.5或SEQ IDNO.6所示。
本发明还提供了一种重组载体，它包括SEQ ID NO.4、SEQ ID NO.5或SEQ ID NO.6所示的核苷酸序列，优选地，所述重组载体是重组pET28a。
本发明还提供了一种重组菌，它包含上述的重组载体；优选地，所述重组菌为重组E.coli。
本发明还提供了一种重组蛋白，其氨基酸序列如SEQ ID NO.1、SEQ ID NO.2或SEQID NO.3所示。
本发明还提供了一种制备上述重组蛋白的方法，它是采用上述的重组菌发酵制得的。
进一步地，所述方法包括以下步骤：
(1)取重组菌，活化，扩增，得种子液；
(2)将种子液接种到培养基中培养，待培养液OD600为0.6～1.0时加入诱导剂诱导表达，收集菌体；所述诱导剂优选为异丙基-β-D-硫代半乳糖苷；
(3)取菌体，破菌，离心，取沉淀纯化后即得。
本发明还提供了一种类贻贝粘蛋白，它是将上述重组蛋白经酪氨酸酶氧化后得到的。
进一步地，它的制备方法包括以下步骤：在包括上述重组蛋白、pH调节剂和抗坏血酸的混合体系中加入酪氨酸酶，反应，提纯，即得。
进一步地，所述混合体系还包括硼酸盐；混合体系中，硼酸盐的浓度为15～25mM，pH调节剂的浓度为0.05～0.15mM，抗坏血酸的浓度为5～15mM；酪氨酸酶的酶活力为10～30U；反应的时间为4～8小时，温度为20～30℃；提纯的方法为：经超滤膜脱盐浓缩；
优选地，所述硼酸盐为硼酸钠，所述pH调节剂为磷酸盐；混合体系中，硼酸盐的浓度为20mM，pH调节剂的浓度为0.1mM，抗坏血酸的浓度为10mM；混合体系的pH为7.0；酪氨酸酶的酶活力为20U；反应的时间为6小时，温度为25℃；所述超滤膜的截留分子量为1000Da。
本发明还提供了上述类贻贝粘蛋白在制备粘附材料中的用途。
本发明提供的类贻贝粘蛋白中间体mlp353，mlp316及mlp319的pI均在10左右，且含有高达23.1％～25.5％摩尔百分数的酪氨酸(Tyr)。mlp353，mlp316及mlp319中，酪氨酸个数及摩尔百分比分别为mlp353:40个(25.5％)；mlp316:52个(24％)；mlp319:64个(23.1％)。
本发明将上述类贻贝粘蛋白中间体mlp353，mlp316及mlp319经酪氨酸酶氧化后得到了新的类贻贝粘蛋白，与天然贻贝粘蛋白相比，本发明的类贻贝粘蛋白具有更高的粘附力和更均匀的分子量分布，与现有技术公开的类贻贝粘蛋白MLAP151相比，本发明的类贻贝粘蛋白也具有更高的粘附力。本发明的类贻贝粘蛋白可以用来制备高粘附力的粘附材料，在海洋工程、生物医学及表面化学等领域具有广阔的应用前景。
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1为E.coli单克隆菌体总蛋白SDS-PAGE结果。(a)图：mlp353；(b)图：mlp316；(c)图：mlp319。
图2为超声破碎提取菌体蛋白SDS-PAGE结果。(a)图：泳道1：mlp353超声前；泳道2：mlp353超声上清；泳道3：mlp353超声沉淀。(b)图：泳道1：对照E.coli Rosseta2(DE3)/pET28a；泳道2：mlp316超声前；泳道3：mlp316超声沉淀；泳道4：mlp316超声上清；泳道5：mlp316超声前；泳道6：mlp319超声沉淀；泳道7：mlp319超声上清。
具体实施方式
本发明所用原料与设备均为已知产品，通过购买市售产品所得。
实施例1：类贻贝粘蛋白中间体的制备
1、构建表达类贻贝粘蛋白中间体的工程菌：E.coli Rosseta2(DE3)/pET28a-mlp353，E.coli Rosseta2(DE3)/pET28a-mlp316，E.coli Rosseta2(DE3)/pET28a-mlp319
分别根据类贻贝粘蛋白中间体mlp353，mlp316及mlp319的氨基酸序列SEQ IDNo.1，SEQ ID No.2及SEQ ID No.3，通过密码子优化获得其核苷酸序列SEQ ID No.4，SEQID No.5，SEQ ID No.6，合成目的基因，并将目的基因片段分别插入到pET28a的多克隆位点Nco I(5’端)和Hind III(3’端)，得到重组表达载体pET28a-mlp353，pET28a-mlp316，pET28a-mlp319和保存重组表达载体的克隆宿主E.coli Top10/pET28a-mlp353，E.coliTop10/pET28a-mlp316，E.coli Top10/pET28a-mlp319。
将pET28a-mlp353，pET28a-mlp316，pET28a-mlp319通过热激法转化感受态细胞E.coli Rosseta2(DE3)，涂布氨苄抗性平板，37℃倒置培养12-16h获得单克隆菌体。具体感受态细胞制备方法和热激转化方法详见《分子克隆实验指南(第3版)》。随机挑取几个单克隆菌体过夜扩增，提取质粒并用Nco I和Hind III进行双酶切验证。琼脂糖凝胶电泳结果显示含有目的基因片段的质粒送出测序，经比对序列正确的单克隆菌体于-20℃保存。
表1.氨基酸序列表
表2.目的基因核苷酸列表
2、mlp353，mlp316，mlp319的重组表达及筛选
种子培养：挑取若干单克隆菌体，接种于含1mL LB培养基的24孔板，并于37℃，700rpm振荡过夜培养。
诱导表达：取样测定种子培养液的OD600，然后吸取20μL新鲜培养的种子液接种于2mL LB培养基，37℃，700rpm振荡培养。待培养液OD600约为0.6-1.0，加入异丙基-β-D-硫代半乳糖苷(IPTG)至终浓度0.4mM，继续培养6h。培养结束后取样进行SDS-PAGE分析，筛选得到表达水平较好的单克隆菌体。
经过对SDS-PAGE结果分析表明(图1)，接种的单克隆菌体均表达了类贻贝粘蛋白中间体，其中，mlp353占总蛋白(整个泳道蛋白条带)的30％，mlp316占总蛋白的45％，mlp319占总蛋白的35％。
3、mlp353，mlp316，mlp319的可溶性分析
种子培养：将E.coli甘油菌液接种于含1mL LB培养基的24孔板，并于37℃，700rpm振荡过夜培养。
诱导表达：取样测定种子培养液的OD600，然后吸取200μL新鲜培养的种子液接种于50mL LB培养基，37℃，700rpm振荡培养。待培养液OD600约为0.6-1.0，加入IPTG至终浓度0.4mM，继续培养6h。
破胞提取：培养结束的培养液离心4000rpm，10min，菌体重悬于5mL的提取缓冲液(20mM Tris-HCl，pH 8.5)。用超声破碎仪进行破胞，工作条件为：功率200W，工作时间3s，间隔时间7s，60次。超声完成后将菌悬液离心12000rpm，5min，上清和沉淀分别取样进行SDS-PAGE分析。
结果如图2所示，经过对超声样品的SDS-PAGE分析表明，超声后mlp353，mlp316，mlp319绝大部分均位于沉淀，说明其在此表达条件下，几乎以不可溶形式于细胞内表达。
4、mlp353，mlp316，mlp319的提取纯化
取700mL的发酵液，6000rpm离心10min后，弃去上清，用1000mL去离子水重悬菌体，经超声破碎后，6000rpm离心，弃去上清，沉淀用500mL的去离子水重悬洗涤，再次离心后，弃去上清，得包涵体沉淀25g。用200mL5％乙酸溶液浸泡、搅拌4h。溶解完全后经8000rpm离心15min后，弃去沉淀杂质，得到含类贻贝粘蛋白中间体的溶液。将溶液pH调至4.0，用去离子水稀释5倍体积后，用CM Sepharose Fast Flow柱进行离子交换层析。先用10倍柱体积的pH4.0柠檬酸-柠檬酸钠缓冲体系平衡层析柱，然后将上述溶液上样吸附。吸附完全后，用2倍柱体积的柠檬酸-柠檬酸钠缓冲体系冲洗，然后用pH 4.0的柠檬酸-柠檬酸钠缓冲液添加0.8M氯化钠的溶液洗脱。洗脱完毕用后用8倍柱体积的0.5M氢氧化钠洗脱。将含类贻贝粘蛋白中间体的洗脱液用截留分子量为1000Da的超滤膜浓缩，用0.5％的乙酸溶液顶洗脱盐，得纯化后的类贻贝粘蛋白中间体溶液。
实施例2：酶催化氧化获得类贻贝粘蛋白MLAP353，MLAP316，MLAP319
将实施例1将含类贻贝粘蛋白中间体(mlp353，mlp316或mlp319)的洗脱液用截留分子量1000Da的超滤膜浓缩至50mL。取硼酸钠、磷酸盐、抗坏血酸加入其中，配制成抗坏血酸终浓度为10mM，硼酸钠终浓度为20mM，磷酸盐终浓度为0.1mM的反应体系，调pH至7.0，加入20U的酪氨酸酶，25℃下缓慢搅拌反应6h。反应完成后用0.5％的乙酸溶液在截留分子量为1000Da的超滤膜中顶洗脱盐后浓缩。将浓缩蛋白溶液冻干，得类贻贝粘蛋白冻干粉：MLAP353，MLAP316，MLAP319。
以下通过实验例证明本发明的有益效果。
实验例1：类贻贝粘蛋白中间体mlp353，mlp316及mlp319的氨基酸组成
(1)
SEQ ID No.1：mlp353；
氨基酸个数：157；
理论分子量：18801.06；
理论等电点(pI)：10.06。
表3.mlp353的氨基酸组成
(2)
SEQ ID No.2：mlp316；
氨基酸个数：217；
理论分子量：25600.93；
理论等电点(pI)：10.04。
表4.mlp316氨基酸组成
| 氨基酸 | 个数 | Mol％ |
| Ala(A) | 8 | 3.70％ |
| Arg(R) | 11 | 5.10％ |
| Asn(N) | 9 | 4.10％ |
| Asp(D) | 2 | 0.90％ |
| Cys(C) | 0 | 0.00％ |
| Gln(Q) | 0 | 0.00％ |
| Glu(E) | 0 | 0.00％ |
| Gly(G) | 37 | 17.10％ |
| His(H) | 4 | 1.80％ |
| Ile(I) | 0 | 0.00％ |
| Leu(L) | 1 | 0.50％ |
| Lys(K) | 41 | 18.90％ |
| Met(M) | 1 | 0.50％ |
| Phe(F) | 0 | 0.00％ |
| Pro(P) | 26 | 12.00％ |
| Ser(S) | 12 | 5.50％ |
| Thr(T) | 7 | 3.20％ |
| Trp(W) | 6 | 2.80％ |
| Tyr(Y) | 52 | 24.00％ |
| Val(V) | 0 | 0.00％ |
(3)
SEQ ID No.3：mlp319；
氨基酸个数：277；
理论分子量：32400.81；
理论等电点(pI)：10.04。
表5.mlp319氨基酸组成
可以看出，类贻贝粘蛋白中间体的pI均在10左右。mlp353，mlp316及mlp319中，酪氨酸(Tyr)个数及摩尔百分比分别为mlp353:40个(25.5％)；mlp316:52个(24％)；mlp319:64个(23.1％)。
实验例2：类贻贝粘蛋白的粘附力测试
1、实验方法
称取实施例2制得的30mg类贻贝粘蛋白冻干粉样品(MLAP353，MLAP316或MLAP319)，放入200μL离心管，加入90μL去离子水超声并搅拌溶解至均一，透亮。取两块金属板(2024t3板材，长*宽*厚：100mm*25mm*1.6mm，两侧打孔)，在下层板涂抹12μL(250平方毫米)，缓慢盖上上层板，然后尽快压上压板(重600g)，至45℃烘箱烘干。48h后将样品取出，移除压板，静置0.5h冷却至室温，将样品用万能拉力机夹具夹住，保证样品长度方向与拉力方向平行。以5±1mm/min速度拉伸，使剪切力变化在8.3-9.8MPa之间。样品断裂后，用最大剪切力计算最大拉伸剪切强度。
以已知的类贻贝粘蛋白MLAP151(即文献Biomaterials 28(2007)3560-3568报道的类贻贝粘蛋白fp-151)为对照样品。
2、实验结果
表6.类贻贝粘蛋白的粘附力测试结果
可以看出，与对照样品MLAP151相比，本发明的类贻贝粘蛋白具有更高的粘附力。
综上，本发明公开了一种粘附力强的类贻贝粘蛋白及其制备方法。本发明提供的类贻贝粘蛋白中间体mlp353，mlp316及mlp319的pI均在10左右，且含有高达23.1％～25.5％摩尔百分数的酪氨酸(Tyr)。本发明将上述类贻贝粘蛋白中间体经酪氨酸酶氧化后得到了新的类贻贝粘蛋白，与天然贻贝粘蛋白相比，本发明的类贻贝粘蛋白具有更高的粘附力和更均匀的分子量分布，与现有技术公开的类贻贝粘蛋白MLAP151相比，本发明的类贻贝粘蛋白也具有更高的粘附力。本发明的类贻贝粘蛋白可以用来制备高粘附力的粘附材料，在海洋工程、生物医学及表面化学等领域具有广阔的应用前景。
SEQUENCE LISTING
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Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro
65 70 75 80
Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro
85 90 95
Ser Tyr Pro Pro Thr Tyr Lys Tyr Asp Gly Tyr Ser Asp Gly Tyr Tyr
100 105 110
Pro Gly Ser Ala Tyr Asn Tyr Pro Ser Gly Ser His Gly Tyr His Gly
115 120 125
His Gly Tyr Lys Gly Lys Tyr Tyr Gly Lys Gly Lys Lys Tyr Tyr Tyr
130 135 140
Lys Tyr Lys Arg Thr Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys
145 150 155 160
Tyr His Arg Lys Gly Tyr Lys Lys Tyr Tyr Gly Gly Gly Ser Ser Ala
165 170 175
Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro
180 185 190
Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro
195 200 205
Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr
210 215 220
Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Gly Gly Asn Tyr Tyr
225 230 235 240
Pro Lys Tyr Lys Tyr Pro Arg Gly Tyr Lys Gly Gly Tyr Asn Gly Tyr
245 250 255
Pro Arg Gly Asn Tyr Gly Trp Asn Lys Gly Trp Lys Lys Gly Arg Trp
260 265 270
Gly Arg Lys Tyr Tyr
275
<210> 4
<211> 471
<212> DNA
<213> 人工序列
<400> 4
atgggtggca attattatcc gaaatataaa tacccgcgtg gctataaagg tggttataat 60
ggctatccgc gtggcaatta tggctggaat aagggttgga aaaaaggtcg ctggggtcgc 120
aaatattatt atgatggtta tagtgacggc tattatccgg gtagcgccta taattatccg 180
agtggtagtc atggctatca tggccacggt tataaaggca aatattatgg caaaggtaaa 240
aagtattact acaagtataa gcgcaccggt aaatataaat atctgaaaaa agcgcgcaag 300
tatcatcgca aaggctataa aaaatactac ggcggcggta gcagtggtgg taattattat 360
cctaaatata agtacccgcg cggctataaa ggcggttata atggttatcc gcgtggtaat 420
tatggttgga ataagggctg gaaaaaaggc cgctggggcc gtaaatatta t 471
<210> 5
<211> 651
<212> DNA
<213> 人工序列
<400> 5
atgggcggta attattatcc gaaatataaa tacccgcgcg gctataaagg cggctataat 60
ggttatccgc gtggtaatta tggttggaat aagggctgga aaaaaggtcg ttggggtcgc 120
aaatattatg caaaaccgag ctatccgccg acctataaag caaaaccgag ttatccgccg 180
acatataaag caaagccgag ttatcctccg acctataagt atgatggtta tagtgatggc 240
tattatccgg gtagtgcata taattatccg agcggtagtc atggttatca tggccacggt 300
tataaaggca aatattatgg caaaggtaaa aagtattact acaagtataa gcgtaccggc 360
aaatataaat atctgaaaaa agcacgtaag taccatcgca aaggctataa aaaatattac 420
ggtggtggca gtagcgcaaa accgtcatat ccgccgacgt ataaagccaa accgagttac 480
ccgccgacct acaaagccaa acctagctat ccgcctacct ataaaggtgg caattattat 540
cctaaataca agtatccgcg tggctataaa ggtggttata atggctatcc gcgtggaaat 600
tatggctgga ataagggttg gaaaaaaggc cgctggggtc gcaagtatta t 651
<210> 6
<211> 834
<212> DNA
<213> 人工序列
<400> 6
atgggtggca attattatcc gaaatataaa tacccgcgcg gctataaagg tggctataat 60
ggttatccgc gtggtaatta tggctggaat aagggctgga aaaaaggccg ttggggtcgc 120
aaatattatg caaaaccgag ctatccgccg acctataaag ccaaaccgag ttatccgccg 180
acatataaag caaaaccgtc atatccgccg acgtataaag ccaagccgag ttatcctccg 240
acctataagg caaaaccgtc ttatccgccg acttataaag ccaaacctag ttatccgcct 300
acctataaat atgatggcta tagcgatggt tattatccgg gtagtgcata taattatccg 360
agtggtagcc acggttatca tggccacggt tataaaggta aatattatgg caaaggtaaa 420
aagtactact acaaatataa gcgcaccggt aaatataaat atctgaaaaa agcccgcaag 480
tatcatcgta aaggttataa aaaatactac ggcggtggca gtagtgccaa accgagctac 540
ccgccgacct acaaagccaa accaagctat ccgcctacat ataaagcgaa accgagttac 600
ccgccgacat acaaagccaa gcctagctat ccgccaacct ataaagcgaa gccgagctat 660
cctccgacat ataaggccaa accgtcttac ccgccgacgt acaaaggcgg taattattat 720
cctaaataca agtatccgcg tggctataaa ggcggttata atggttaccc gcgtggtaac 780
tatggttgga ataagggttg gaaaaaaggt cgctggggtc gcaagtatta ttaa 834
Claims (14)
1.一种基因，其特征在于：其核苷酸序列如SEQ ID NO.4、SEQ ID NO.5或SEQ ID NO.6所示。
2.一种重组载体，其特征在于：它包括SEQ ID NO.4、SEQ ID NO.5或SEQ ID NO.6所示的核苷酸序列。
3.根据权利要求2所述的重组载体，其特征在于：所述重组载体是重组pET28a。
4.一种重组菌，其特征在于：它包含权利要求2或3所述的重组载体。
5.根据权利要求4所述的重组菌，其特征在于：所述重组菌为重组E.coli。
6.一种重组蛋白，其特征在于：其氨基酸序列如SEQ ID NO.1、SEQ ID NO.2或SEQ IDNO.3所示。
7.一种制备权利要求6所述重组蛋白的方法，其特征在于：它是采用权利要求4或5所述的重组菌发酵制得的。
8.根据权利要求7所述的方法，其特征在于：所述方法包括以下步骤：
(1)取重组菌，活化，扩增，得种子液；
(2)将种子液接种到培养基中培养，待培养液OD600为0.6～1.0时加入诱导剂诱导表达，收集菌体；
(3)取菌体，破菌，离心，取沉淀纯化后即得。
9.根据权利要求8所述的方法，其特征在于：所述诱导剂为异丙基-β-D-硫代半乳糖苷。
10.一种类贻贝粘蛋白，其特征在于：它是将权利要求6所述重组蛋白经酪氨酸酶氧化后得到的。
11.根据权利要求10所述的类贻贝粘蛋白，其特征在于：它的制备方法包括以下步骤：在包括权利要求6所述重组蛋白、pH调节剂和抗坏血酸的混合体系中加入酪氨酸酶，反应，提纯，即得。
12.根据权利要求11所述的类贻贝粘蛋白，其特征在于：所述混合体系还包括硼酸盐；混合体系中，硼酸盐的浓度为15～25mM，pH调节剂的浓度为0.05～0.15mM，抗坏血酸的浓度为5～15mM；酪氨酸酶的酶活力为10～30U；反应的时间为4～8小时，温度为20～30℃；提纯的方法为：经超滤膜脱盐浓缩。
13.根据权利要求12所述的类贻贝粘蛋白，其特征在于：所述硼酸盐为硼酸钠，所述pH调节剂为磷酸盐；混合体系中，硼酸盐的浓度为20mM，pH调节剂的浓度为0.1mM，抗坏血酸的浓度为10mM；混合体系的pH为7.0；酪氨酸酶的酶活力为20U；反应的时间为6小时，温度为25℃；所述超滤膜的截留分子量为1000Da。
14.权利要求10～13任一项所述类贻贝粘蛋白在制备粘附材料中的用途。
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| CN1989152A (zh) * | 2004-03-26 | 2007-06-27 | Posco公司 | 贝类生物粘合剂 |
| CN108503712A (zh) * | 2018-03-14 | 2018-09-07 | 天津大学 | 具有粘附-抗污双功能的贻贝粘附蛋白/两性离子多肽融合蛋白及合成方法 |
| CN112946041A (zh) * | 2021-02-06 | 2021-06-11 | 自然资源部第一海洋研究所 | 一种基于融合蛋白传感器的重金属离子检测方法 |
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| CN1989152A (zh) * | 2004-03-26 | 2007-06-27 | Posco公司 | 贝类生物粘合剂 |
| CN108503712A (zh) * | 2018-03-14 | 2018-09-07 | 天津大学 | 具有粘附-抗污双功能的贻贝粘附蛋白/两性离子多肽融合蛋白及合成方法 |
| CN112946041A (zh) * | 2021-02-06 | 2021-06-11 | 自然资源部第一海洋研究所 | 一种基于融合蛋白传感器的重金属离子检测方法 |
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