CN116396403B - 一种重组纤维连接蛋白功能性短体及其制备方法 - Google Patents

一种重组纤维连接蛋白功能性短体及其制备方法 Download PDF

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CN116396403B
CN116396403B CN202310364316.8A CN202310364316A CN116396403B CN 116396403 B CN116396403 B CN 116396403B CN 202310364316 A CN202310364316 A CN 202310364316A CN 116396403 B CN116396403 B CN 116396403B
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fibronectin
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peptide
recombinant fibronectin
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CN116396403A (zh
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邱雪
何晓燕
王璐
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Guangdong Zhongyu Biotechnology Co ltd
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Abstract

本发明公开了一种重组纤维连接蛋白功能性短体及其制备方法。重组纤维连接蛋白功能性短体包括细胞结合肽、纤维连接蛋白胶原蛋白结合蛋白结合域序列和金环蛇毒抗菌肽顺序连接的融合多肽；所述融合肽的氨基酸序列如SEQ ID NO.1所示的氨基酸序列；所述细胞结合肽的氨基酸序列如SEQ ID NO.2所示的氨基酸序列；所述纤维连接蛋白结合胶原蛋白的结构域序列的氨基酸序列如SEQ ID NO.3所示的氨基酸序列；所述金环蛇毒抗菌肽的氨基酸序列如SEQ ID NO.4所示的氨基酸序列。本发明重组纤维连接蛋白功能性短体及其制备方法，能有效解决纤维连接蛋白分子量小，半衰期短，使用时容易红肿感染的缺点，有利于在促细胞吸附和生长、伤口愈合和抗菌方面的应用。

Description

一种重组纤维连接蛋白功能性短体及其制备方法
技术领域
本发明涉及生物制药领域，具体涉及了一种重组纤维连接蛋白功能性短体及其制备方法。
背景技术
纤维连接蛋白是一种大的纤维状糖蛋白，由两个亚基通过C末端的二硫键交联形成，每个亚基的分子量为220～250kDa，整个分子呈V型，广泛分布于机体组织中。传统的纤维连接蛋白提取自动物血浆，具有一定的风险性，且其分子量较大，难以渗透进入皮肤被吸收利用。如中国发明专利CN102675473A公开了一种基因重组人型活性碱性成纤维细胞生长因子融合蛋白，该融合蛋白全长762个氨基酸，氮端为Ⅰ型人胶原蛋白500-1099肽段共600个氨基酸，碳端为人碱性成纤维细胞因子共154个氨基酸，两个肽段之间用谷氨酸和苯丙氨酸以及一个含6个氨基酸的柔性肽段连接；目前重组的纤维连接蛋白主要取其功能结构区一部分，分子量大大减少，一般为60～70kD，但仍存在渗透力差，作用单一的缺点。特别是应用到美容领域时，为增加蛋白液渗透率，通常采用进一步减小重组纤维蛋白分子量或微针微创的方法。但是采用更小分子量的短肽，一方面其表达后易降解，半衰期短，另一方面纯化工艺困难。而采用微针微创的方法常常会引起细菌感染，造成皮肤的红肿甚至溃烂。而在应用到损伤治疗领域时，也需要格外注意感染情况的发生。
发明内容
为解决现有技术的不足，本发明的目的在于提供了一种重组纤维连接蛋白功能性短体及其应用，能有效解决皮肤渗透能力及吸附能力差、作用时间短、易感染的技术问题。
为实现上述目的，本发明第一方面提供一种重组纤维连接蛋白功能性短体，具体为细胞结合肽、连接肽、纤维连接蛋白结合胶原蛋白的结构域序列、连接肽和金环蛇毒抗菌肽几种活性多肽顺序连接的融合多肽；
所述融合肽的氨基酸序列如SEQ ID NO.1所示的氨基酸序列；
所述细胞结合肽的氨基酸序列如SEQ ID NO.2所示的氨基酸序列；
所述纤维连接蛋白结合胶原蛋白的结构域序列的氨基酸序列如SEQ ID NO.3所示的氨基酸序列；
所述金环蛇毒抗菌肽的氨基酸序列如SEQ ID NO.4所示的氨基酸序列。
金环蛇是一种广泛分布于我国广东、广西、福建、江西、海南及云南地区的具有前沟牙的毒蛇。其体内的抗菌肽具有广谱的抗菌活性，对革兰氏阳性菌、革兰氏阴性菌、某些真菌及病毒均具有非常强的杀伤活性，对一些耐药性细菌同样具有作用。
进一步地，在本发明中，所述连接肽的氨基酸序列为GGGGSNNKGGGGS或GGGGSNNKNNKGGGGS或GGGGSNNKNNKNNKGGGGS中的一种。
本发明第二方面，提供了一种编码上述重组纤维连接蛋白功能性短体蛋白的核苷酸序列，所述核苷酸序列如SEQ ID NO.5所示。
本发明第三方面，提供了一种重组表达载体pGAPZa-A FN，所述表达载体包括SEQID NO.1所示的氨基酸序列。
本发明第四方面，提供了一种酵母工程菌，含有所述的重组表达载体。
本发明第五方面，一种重组纤维连接蛋白功能性短体的制备方法，包括如下步骤：
(1)在如SEQ ID NO.5所示核苷酸序列5’和3’两端加入限制性内切酶核苷酸序列；
(2)将步骤(1)序列插入到表达载体pGAPZa-A上，并线性化，转入毕赤酵母感受态细胞中，筛选高表达重组纤维连接蛋白功能性短体的单克隆菌株；
(3)将步骤(2)筛选的菌株进行表达纯化，获得重组纤维连接蛋白功能性短体。
本发明第六方面，本发明所述重组纤维连接蛋白功能性短体在细胞接触吸附和伸展延伸方面的应用，具体如在细胞培养中的应用。
本发明第七方面，本发明所述重组纤维连接蛋白功能性短体在伤口愈合方面的应用，具体如在组织缝合中的应用。
本发明第八方面，本发明所述重组纤维连接蛋白功能性短体在抗菌方面的应用，具体如在制备抗菌材料中的应用。
本发明的有益效果：
1、本发明的细胞结合肽与纤维连接蛋白结合胶原蛋白结合域序列、蛇毒抗菌肽，通过富含天冬酰胺和赖氨酸的柔性连接肽相连接，得到增强了与细胞、胶原蛋白结合的能力，并同时具备抗菌活性，可有效解决微创易感染的问题。
2、本发明的重组纤维连接蛋白功能性短体分子量更小，约42kD，皮肤渗透率较传统的重组纤维连接蛋白(60～70kD)更佳。且通过融合细胞结合肽和柔性连接肽解决了单一纤维连接蛋白功能结构域半衰期短(3min)的缺点，蛋白更稳定，有效作用时间长(半衰期延长至大于20h)。
3、本发明构建的重组纤维连接蛋白功能性短体应用广泛，能够应用于化妆品添加剂，临床上皮肤损伤修复等方面。
附图说明
图1为商用载体pGAPZa-A；
图2为本发明高拷贝毕赤酵母菌株的筛选平板，其中1～23为高浓度抗生素筛选出的单克隆划线培养；
图3为本发明高表达菌株的电泳结果图，其中泳道1为毕赤酵母空宿主上清液sds-page电泳图，图2至6分别对应图2中4/9/11/13/14号克隆子发酵上清sds-page电泳图；
图4为本发明图3高表达菌株的蛋白免疫印迹结果图，其中1为毕赤酵母空宿主上清组，图2至6分别对应图2中4/9/11/13/14号克隆子发酵上清组；
图5为本发明为重组纤维连接蛋白的纯化图谱，其中泳道1～3分别为弱阳离子交换柱层析纯化，疏水层析纯化，分子筛层析纯化收集液sds-page电泳图；
图6为本发明不同浓度重组纤维连接蛋白促细胞吸附曲线图；
图7为本发明不同浓度重组纤维连接蛋白促细胞增殖曲线图；
图8为本发明不同浓度重组纤维连接蛋白对痤疮杆菌的抑制作用图，其中1～6分别是不同浓度的重组纤维连接蛋白功能性短肽对痤疮杆菌的抑制效果；
图9为未融合金环蛇抗菌肽的细胞结合肽、连接肽、纤维连接蛋白结合胶原蛋白结构域序列顺序连接融合蛋白纯化后sds-page电泳图；
图10为不同浓度重组纤维连接蛋白及细胞结合肽、连接肽、纤维连接蛋白结合胶原蛋白结构域序列顺序连接融合蛋白不同促细胞增殖曲线图。
具体实施方式
如背景技术描述的，目前重组的纤维连接蛋白主要取其功能结构区一部分，分子量大大减少，一般为60～70kD，但仍存在渗透力差，作用单一的缺点。特别是应用到美容领域时，为增加蛋白液渗透率，通常采用进一步减小重组纤维蛋白分子量或微针微创的方法。但是采用更小分子量的短肽，一方面其表达后易降解，半衰期短，纯化工艺困难。而采用微针微创的方法常常会引起细菌感染，造成皮肤的红肿甚至溃烂。而在应用到损伤治疗领域时，也需要格外注意感染情况的发生。
为了解决上述技术问题，本发明提出了一种重组纤维连接蛋白功能性短体，具体为细胞结合肽、纤维连接蛋白结合胶原蛋白的结构域序列和金环蛇毒抗菌肽三种活性多肽通过连接肽顺序连接的融合多肽；
所述融合肽的氨基酸序列如SEQ ID NO.1所示的氨基酸序列；
所述细胞结合肽的氨基酸序列如SEQ ID NO.2所示的氨基酸序列；
所述纤维连接蛋白结合胶原蛋白的结构域序列的氨基酸序列如SEQ ID NO.3所示的氨基酸序列；
所述金环蛇毒抗菌肽的氨基酸序列如SEQ ID NO.4所示的氨基酸序列。
金环蛇是一种广泛分布于我国广东、广西、福建、江西、海南及云南地区的具有前沟牙的毒蛇。其体内的抗菌肽具有广谱的抗菌活性，对革兰氏阳性菌、革兰氏阴性菌、某些真菌及病毒均具有非常强的杀伤活性，对一些耐药性细菌同样具有作用。
本发明的细胞结合肽与纤维连接蛋白结合胶原蛋白结合域序列、蛇毒抗菌肽，通过富含天冬酰胺和赖氨酸的柔性连接肽相连接，得到增强了与细胞、胶原蛋白结合的能力，并同时具备抗菌活性，可有效解决微创易感染的问题。本发明的重组纤维连接蛋白功能性短体分子量更小，约42kD，皮肤渗透率较传统的重组纤维连接蛋白(60～70kD)更佳。且通过融合细胞结合肽和柔性连接肽解决了单一纤维连接蛋白功能结构域半衰期短(3min)的缺点，蛋白更稳定，有效作用时间长(半衰期延长至大于20h)。
以下结合具体实施例，对本发明做进一步说明。所描述实施例是本申请一部分实施例，而不是全部实施例。基于本申请中的实施例，本领域普通技术人员在没有创造性劳动前提下所获得的所有其他实施例，都属于本申请的保护范围。
实施例1构建编码pGAPZa-A FN蛋白的表达质粒
本实施例中使用的商用载体pGAPZa-A(如图1所示)，购自Invitrogen公司，根据图1相关位置设计酶切位点xhoI和xbaI。通过毕赤酵母偏爱密码子人工优化获得编码重组纤维纤维连接蛋白功能性短体的核苷酸序列，经人工合成获得。所合成获得的核苷酸序列5’和3’端分别含有xhoI和xbaI的酶切位点，重组纤维连接蛋白功能性短体的核苷酸片段将插入在pGAPZa-A同样的酶切位点之间，获得编码pGAPZa-A FN蛋白的表达质粒，其中重组纤维连接蛋白功能性短体的氨基酸序列如下：
PHSRNRGDDSGVVYSVGMQWLKTQGNKQMLCTCLGNGVSCQETAVTQTYGGNSNGE
PCVLPFTYNGRTFYSCTTEGRQDGHLWCSTTSNYEQDQKYSFCTDHTVLVQTRGGNSNGALCH
FPFLYNNHNYTDCTSEGRRDNMKWCGTTQNYDADQKFGFCPMAAHEEICTTNEGVMYRIGD
QWDKQHDMGHMMRCTCVGNGRGEWTCIAYSQLRDQCIVDDITYNVNDTFHKRHEEGHML
NCTCFGQGRGRWKCDPVDQCQDSETGTFYQIGDSWEKYVHGVRYQCYCYGRGIGEWHCQPL
GGGGSNNKNNKNNKGGGGSPHSRNRGDGGGGSNNKNNKNNKGGGGSKFFRKLKKSVKKRA
KEFFKKPRVIGVSIPF(SEQ ID NO.1)。
重组纤维连接蛋白功能性短体具体包括如下氨基酸序列：
(1)细胞结合肽的氨基酸序列如SEQ ID NO.2所示；SEQ ID NO.2：PHSRNRGDGHCVT；
(2)纤维连接蛋白胶原结合蛋白结构域氨基酸序列如SEQ ID NO.3所示；
SEQ ID NO.3：
DSGVVYSVGMQWLKTQGNKQMLCTCLGNGVSCQETAVTQTYGGNSNGEPCVLPFTYNGRTF
YSCTTEGRQDGHLWCSTTSNYEQDQKYSFCTDHTVLVQTRGGNSNGALCHFPFLYNNHNYTDC
TSEGRRDNMKWCGTTQNYDADQKFGFCPMAAHEEICTTNEGVMYRIGDQWDKQHDMGH
MMRCTCVGNGRGEWTCIAYSQLRDQCIVDDITYNVNDTFHKRHEEGHMLNCTCFGQGRGRWKCDPVDQCQDSETGTFYQIGDSWEKYVHGVRYQCYCYGRGIGEWHCQPL；
(3)金环蛇毒抗菌肽氨基酸序列如SEQ ID NO.4所示；SEQ ID NO.4：KFFRKLKKSVKKRAKEFFKKPRVIGVSIPF。
重组纤维连接蛋白功能性短体的核苷酸序列SEQ ID NO.5所示，具体如下：
1 CCACATTCAC GTAATCGGGG TGATGATAGC GGGGTCGTCT ATTCCGTGGG AATGCAGTGG
61 TTAAAGACGC AGGGTAATAA ACAAATGCTC TGTACATGTT TGGGTAATGG TGTGTCGTGT
121 CAGGAGACCG CAGTTACACA AACCTATGGA GGGAACTCGA ATGGGGAGCC TTGCGTACTT
181 CCCTTTACGT ACAACGGCAG GACATTTTAC TCCTGTACGA CTGAGGGTCG ACAAGATGGT
241 CATCTGTGGT GCTCCACTAC TAGCAATTAC GAACAAGACC AAAAATATTC TTTCTGTACG
301 GACCATACGG TGCTAGTCCA GACTCGCGGG GGCAATTCCA ACGGGGCTCT TTGCCACTTC
361 CCTTTCTTAT ATAACAATCA TAACTATACT GACTGTACAT CTGAGGGTCG GCGCGACAAC
421 ATGAAGTGGT GCGGAACCAC CCAAAACTAC GACGCGGATC AGAAGTTTGG CTTCTGCCCA
481 ATGGCTGCCC ATGAAGAGAT ATGCACCACG AATGAAGGGG TTATGTATCG AATCGGGGAT
541 CAATGGGATA AACAGCACGA CATGGGACAT ATGATGCGGT GCACCTGTGT GGGTAATGGC
601 CGTGGTGAAT GGACCTGCAT TGCATACAGC CAGCTGAGAG ACCAATGCAT TGTAGATGAC
661 ATCACTTACA ATGTCAACGA CACATTTCAC AAACGACACG AAGAGGGCCA CATGTTGAAT
721 TGCACTTGTT TCGGACAAGG TCGGGGCCGC TGGAAGTGTG ACCCGGTTGA TCAGTGTCAG
781 GATAGTGAGA CAGGAACGTT TTACCAAATA GGAGACTCAT GGGAGAAGTA CGTACACGGG
841 GTTAGATATC AGTGTTACTG TTATGGCAGG GGCATCGGGG AATGGCATTG CCAACCACTC
901 GGCGGAGGGG GATCAAACAA TAAGAATAAC AAAAACAACA AAGGAGGAGG AGGTAGTCCG
961 CACTCGCGCA ACAGAGGGGA TGGCGGGGGG GGTAGTAACA ACAAGAATAA TAAGAATAAC
1021 AAGGGTGGCG GCGGATCAAA ATTTTTCCGT AAACTAAAGA AGAGCGTTAA AAAAAGGGCC
1081 AAAGAATTCT TTAAGAAACC CAGAGTAATA GGCGTGTCTA TTCCCTTT
(SEQ ID NO.5)
重组纤维连接蛋白功能性短体委托南京金斯瑞生物科技有限公司进行合成。
实施例2重组纤维连接蛋白功能性短体的表达纯化
(1)高克隆高表达单克隆菌株的筛选：提取表达载体pGAPZa-A FN，使其浓度达到1μg/ml，用Hind III限制性内切酶进行单酶切，取5～10μl质粒与100μl毕赤酵母SMD1168感受态细胞进行混合，放入预冷的电转杯(0.1cm)中，冰上放置10min。设置电转参数：脉冲电压为500V，脉冲宽度15ms，脉冲次数2次后，立即加入1ml预冷的1M山梨醇，每200μl涂一个含25μg/ml博纳霉素(zeocin)YPDS培养基平板，置于30℃培养箱，培养48～60h。挑选边缘光滑，颜色乳白、个体较大的单克隆菌落，采用划线法置于含100μg/ml博纳霉素(zeocin)YPDS培养基平板，置于30℃培养箱，培养48～60h。继续挑选边缘光滑，颜色乳白、个体较大的单克隆菌落，采用划线法置于含250μg/ml博纳霉素(zeocin)YPD培养基平板，置于30℃培养箱，培养48～60h。继续挑选边缘光滑，颜色乳白、个体较大的单克隆菌落，采用划线法置于含500μg/ml博纳霉素(zeocin)YPD培养基平板，置于30℃培养箱，培养48～60h，不同浓度博纳霉素YPD平板筛选见图2。从含500μg/ml博纳霉素(zeocin)YPD培养基平板上挑出的菌落，转入含100μg/ml博纳霉素(zeocin)YPD液体培养基(3ml)中，30℃，200rpm振荡培养48～60h，10000rpm离心10min，取上清进行sds-page电泳及western blot来分析上清的蛋白表达情况，结果结果见图3及图4。
(2)高表达菌株的高密度发酵及纯化：挑选高表达菌株，按接种密度5％，采用30％临界溶氧法进行高密度发酵。即当发酵罐内溶氧从100％下降至30％以下时，停止补加碳源(20％蔗糖)，当溶氧升至30％以上时，补加碳源，设置溶氧与补料及转速相关联。待补加碳源不能降低溶氧，且罐内pH值上升，即补酸显著增加时，停止发酵。4000rpm，离心30分钟，收集上清。上清液再经1.0及0.65μm滤芯二级过滤，得到发酵澄清处理液。上清与浓度为200mmol/L的磷酸缓冲液(pH7.5)按9:1的体积比进行混匀。经弱阳离子交换柱吸附后，再用含300mmol/L氯化钠，20mmol/L磷酸盐缓冲液，pH7.5进行洗脱。将洗脱下来的收集液，补加硫酸铵至终浓度为1.5mol/L，采用梯度洗脱的方法，采用疏水凝胶层析的方法进行纯化，收集液经脱盐柱G25置换为10mmol/L磷酸盐缓冲液，pH7.5。所获得的蛋白纯度经SDS-PAGE电泳鉴定，无降解条带，纯度大于95％，见图5。
实施例3重组纤维连接蛋白功能性短体的促细胞吸附作用
将实施例2纯化得到的蛋白样品稀释至蛋白浓度为2.5/5/10/20/40μg/ml后，包被至96孔板1h后，用PBS洗涤2遍。加入5％的BSA封闭1h后，加入永生胚胎成纤维细胞(3T3细胞)，置于37℃，5％CO2培养1h，吸取孔中培养基，加入PBS轻轻漂洗1～2次。对照组为10mmol/L的PBS。用CCK8活细胞检测试剂盒检测活细胞数量，验证本重组纤维连接蛋白功能性短体的活性，结果如图6。由图6可知重组纤维连接蛋白功能性短体有较好的细胞促吸附作用，且加入了重组纤维连接蛋白功能性短体的组别细胞更为细长。
实施例4重组纤维连接蛋白功能性短体的促细胞增殖作用
将实施例2纯化得到的蛋白样品稀释至蛋白浓度为2.5/5/10/20/40μg/ml后，包被至96孔板1h后，加入4000个/孔的永生胚胎成纤维细胞(3T3细胞)，置于37℃，5％CO2培养72h。弃去上清，用PBS轻轻漂洗两次，采用CCK8法检测活细胞数量，绘制不同浓度样品增殖曲线，如图7，计算半数有效量ED50。结果表明，重组纤维连接蛋白功能性短体的半数有效量ED50为8.518μg/ml，表明我们发明的重组纤维连接蛋白功能性短体能有效的促进细胞的增殖。
实施例5重组纤维连接蛋白功能性短体的抑菌作用
采取厌氧菌痤疮杆菌作为试验对象，采用透明斑点法检测重组纤维连接蛋白功能性短体的抑菌活性。即，将实施例2获得的将活化后的痤疮杆菌脑心浸液固体培养基中，取20ul不同浓度样品滴加至合适大小的无菌圆形滤纸上，放入含厌氧袋的密封袋中，37℃培养过夜，观察抑制圈大小，如图8。由图8可知本重组纤维连接蛋白功能性短体具有较好的抑菌效果。
对比例1
采用实施例2的方法，制备细胞结合肽、连接肽、纤维连接蛋白结合胶原蛋白结构域序列顺序连接融合的纯化蛋白，即无蛇毒抗菌肽融合蛋白，如图9，将纯化蛋白稀释至蛋白浓度为2.5/5/10/20/40μg/ml后，包被至96孔板1h后，用PBS洗涤2遍。加入5％的BSA封闭1h后，加入永生胚胎成纤维细胞(3T3细胞)，置于37℃，5％CO2培养1h，吸取孔中培养基，加入PBS轻轻漂洗1～2次。对照组为10mmol/L的PBS。用CCK8活细胞检测试剂盒检测活细胞数量，结果如图10所示。由图10可知细胞结合肽、连接肽、纤维连接蛋白结合胶原蛋白结构域序列顺序连接融合的蛋白，同样具有促细胞吸附增殖作用，但细胞增殖效果较实施例4差。
上述稀释后各蛋白按实施例5进行痤疮杆菌的抑制试验，并未观察到明显的抑菌圈。
功能性短体有较好的细胞促吸附作用，且加入了重组纤维连接蛋白功能性短体的组别细胞更为细长，表明其细胞伸展性更好。
综上所述，本发明的重组连接蛋白功能性短肽不仅分子量更小，半衰期长，而且具有较好的促细胞吸附、增殖作用，同时具备优秀的抑菌作用，能较好的应用于美容化妆品添加剂，临床上皮肤损伤修复等领域。
以上所述实施例仅表达了本发明的实施方式，其描述较为具体和详细，但并不能因此而理解为对本实用新型专利范围的限制，但凡采用等同替换或等效变换的形式所获得的技术方案，均应落在本实用新型的保护范围之内。

Claims (5)

1.一种重组纤维连接蛋白功能性短体，其特征在于，其为细胞结合肽、纤维连接蛋白结合胶原蛋白的结构域序列、蛇毒抗菌肽顺序连接的融合肽；
所述重组纤维连接蛋白功能性短体的氨基酸序列如SEQ ID NO.1所示。
2.一种编码如权利要求1所述的重组纤维连接蛋白功能性短体的核苷酸序列，其特征在于，所述核苷酸序列如SEQ ID NO.5所示。
3.一种重组表达载体pGAPZa-A FN，其特征在于，所述表达载体包括权利要求2所述的核苷酸序列。
4.一种酵母工程菌，其特征在于，含有权利要求3所述的重组表达载体。
5.权利要求1所述的重组纤维连接蛋白功能性短体的制备方法，其特征在于，包括以下步骤：
（1）在如SEQ ID NO.5所示核苷酸序列5’和3’两端加入限制性内切酶核苷酸序列；
（2）将步骤（1）序列插入到表达载体pGAPZa-A上，并线性化，转入毕赤酵母感受态细胞中，筛选高表达重组纤维连接蛋白功能性短体的单克隆菌株；
（3）将步骤（2）筛选的菌株进行表达纯化，获得重组纤维连接蛋白功能性短体。
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