CN116855434A - 一种重组大肠杆菌及其在制备酪氨酸血症药物中的应用 - Google Patents
一种重组大肠杆菌及其在制备酪氨酸血症药物中的应用 Download PDFInfo
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Abstract
本发明公开了一种重组大肠杆菌及其在制备酪氨酸血症药物中的应用,属于生物技术领域。本发明通过酪氨酸解氨酶的筛选及蛋白标签的添加构建了一株重组工程菌株,具体地,利用基因查找的手段,收集自然界中具有酪氨酸降解活性的酶分子,并对其酪氨酸降解活性进行体外活性检测,筛选得到具有优异活性的酶分子,将筛选出的酶分子转入大肠杆菌中进一步选出高表达酪氨酸解氨酶分子且具有高效降解酪氨酸能力的工程菌,然后为提高降解活性引入蛋白标签,发现其对肠胃液的耐受性也有所提高,有望用于制备酪氨酸血症治疗药物。
Description
技术领域
本发明涉及一种重组大肠杆菌及其在制备酪氨酸血症药物中的应用,属于生物技术领域。
背景技术
酪氨酸血症(tyrosinemia)是由于酪氨酸代谢途径中的酶缺陷,引起的血浆中酪氨酸浓度增高,不同步骤的酶的缺陷可导致多种临床表现不同的疾病,如导致脑、肝、肾、骨骼等多脏器损害,预后不良,致死及致残率很高。酪氨酸在人体的来源包括饮食摄入和内源性合成两部分,内源性生成的底物来源为食源性的必需氨基酸酪氨酸。酪氨酸血症分为3种类型。
酪氨酸血症Ⅰ型,也被称为肝肾酪氨酸血症(hepatorenal tyrosinemia,HT-1),为延胡索酰乙酰乙酸水解酶(fumarylacetoacetate hydrolase,FAH)缺陷所致,以肝、肾和周围神经病变为特征。HT-1影响男性和女性的数量相等,总体发病率为1/2000~1/100000。美国人群的突变携带频率1/150~1/100,由于奠基者效应,斯堪的纳维亚半岛HT-1型的活产新生儿发病率约为1/74 000,芬兰和挪威约为1/60 000。此外,在加拿大的魁北克省,活产新生儿发病率约为1/16 000,携带频率约为1/66。据估计,魁北克省Saguenay-Lac Saint-Jean地区的新生儿患病率为1/1850。根据HT-1患者发病年龄,临床上将该疾病分为急性型(发病年龄<2个月)、亚急性型(发病年龄2-6个月)和慢性型(发病年龄>6个月)。急性患儿多在出生后2个月内急性发病,病情迅速恶化,常在出生后第3-9月死于肝功能衰竭。亚急性型与急性型相似,但症状出现在出生后2-6个月之间。慢性型发病时间通常在出生6个月之后,临床表现以渐进性肝和肾损害为主,随着病情的发展最终会导致肝硬化和Fanconi综合征,甚至出现佝偻病。FAH酶缺乏会导致有毒代谢物的积累,包括延胡索酰乙酰乙酸(FAA)和马来酸乙酰乙酸(maleylacetoacetate)。由于其上游代谢物酪氨酸(tyrosine)、4-羟基苯丙酮酸(4-hydroxyphenylpyruvate)含量升高,使中间代谢产物马来酰乙酰乙酸进一步积累,从而刺激产生旁代谢通路,导致其代谢产物琥珀酰乙酰乙酸(succinylacetoacetate)、琥珀酰丙酮增高(succinylacetone,SA)。这两种旁代谢产物可以与蛋白质的巯基结合,是造成肝肾损害的主要原因。当不接受治疗时,大多数HT-1患者在婴儿早期就会死于急性严重肝肾功能衰竭。酪氨酸血症Ⅱ型,为酪氨酸氨基转移酶(tyrosine aminotransferase,TAT)缺陷所致,以角膜增厚、掌跖角化和发育落后为特征。酪氨酸血症Ⅲ型,极为罕见,为4-羟基苯丙酮酸双加氧酶(hydroxyphenylpyruvic acid dioxygenase,HPPD)缺陷所致,以神经精神症状为主要表现。
目前针对酪氨酸血症存在唯一一款上市药物——尼替西农(NTBC)。NTBC是4-羟基苯丙酮酸双加氧酶的竞争性抑制剂,可抑制HT-1患者体内酪氨酸的常规分解代谢,阻止毒性代谢产物在体内蓄积,从而避免肝肾功能损害,提高患者存活率。患者在尼替西农治疗期间必须限制饮食中酪氨酸和酪氨酸摄入量。研究中报告的常见不良反应为血小板减少、白细胞减少和视觉***病症,包括结膜炎、角膜混浊、角膜炎和畏光。但由于NTBC为4-羟基苯丙酮酸双加氧酶抑制剂,抑制了酪氨酸向4-羟基苯丙酮酸的代谢,故血酪氨酸增高,若血酪氨酸水平超过500μmol/L,需要控制蛋白质摄入,给予不含酪氨酸、酪氨酸配方营养粉饮食治疗,否则会由于血酪氨酸水平过高导致角膜损伤。
其他HT-1的治疗方法包括肝移植及基因治疗。肝移植治疗HT-1已有20多年历史,可显著改善患者的肝脏、肾脏及神经***症状。近几年随着NTBC的应用需要肝移植者逐渐减少。另外,肝移植术后患儿仍有约5%-10%的病死率,且术后仍需终生进行免疫抑制治疗,且此方法受手术条件及供体数量少的限制,临床上应用存在困难,故只有当患儿肝功能严重衰竭,且对尼替西农治疗无效或由于其他原因得不到尼替西农治疗及肝组织有发生恶变的依据时,选择肝移植治疗。至于基因治疗目前仍处于动物研究阶段。
本发明旨在通过开发活体微生物药物从食源性代谢的角度对此疾病进行治疗,目前尚未有相关的报道。
发明内容
为解决上述问题,本发明提供了一种重组大肠杆菌,通过引入来源于约氏黄杆菌(Flavobacterium johnsoniae)的酪氨酸解氨酶FjTAL实现全菌体的降解活性保持,并进一步融合表达蛋白标签,筛选得到酪氨酸降解效果最优的结构。同时对不同菌株进行肠胃液耐受性实验,发现其中一株工程菌在耐受实验中表现最为优异,为制备酪氨酸血症治疗药物奠定了基础。
本发明的第一个目的是提供一种表达酪氨酸解氨酶的重组大肠杆菌,其异源表达了氨基酸序列如SEQ ID NO.1所示的酪氨酸解氨酶FjTAL,并在酪氨酸解氨酶FjTAL的N端融合表达了蛋白标签。
进一步地,蛋白标签选自SUMO标签、GST标签或GroE标签。
进一步地,SUMO标签的核苷酸序列如SEQ ID NO.8所示;GST标签的核苷酸序列如SEQ ID NO.9所示;GroE标签的核苷酸序列如SEQ ID NO.10所示。
进一步地,酪氨酸解氨酶FjTAL的核苷酸序列如SEQ ID NO.7所示。
进一步地,以Escherichia coli Nissle1917(EcN1917)为出发菌株。
进一步地,以pGEX-4T-2为表达载体。
本发明的第二个目的是提供上述重组大肠杆菌的构建方法,包括以下步骤:
S1、将蛋白标签融合至酪氨酸解氨酶FjTAL的N端,并连接至表达载体上,得到重组质粒;
S2、将S1得到的重组质粒导入大肠杆菌宿主中,得到上述重组大肠杆菌。
本发明的第三个目的是提供上述重组大肠杆菌在降解酪氨酸中的应用,如制备成降解酪氨酸的产品用于体外或体内降解酪氨酸。
本发明的第四个目的是提供上述重组大肠杆菌在制备酪氨酸血症预防或治疗药物中的应用。
本发明的第五个目的是提供一种可降解酪氨酸的微生物菌剂,含有上述重组大肠杆菌。
进一步地,所述微生物菌剂为液态菌剂或固态菌剂。
本发明的有益效果:
(1)本发明提供了一株体外和体内酶活均较高的重组大肠杆菌,该工程菌株融合表达了蛋白标签和酪氨酸解氨酶FjTAL,不仅可以高效表达酪氨酸解氨酶,而且表现出显著高于其他酪氨酸解氨酶的酪氨酸降解活性,从而可以有效地降解酪氨酸,避免其在体内蓄积。
(2)目前常见的治疗酪氨酸血症的药物大多是化学药物,存在较高的毒副作用、容易产生耐药性等缺点;PEG修饰药物、口服蛋白制剂药物亦存在免疫排斥反应及不稳定等弊端。而本发明采用毒副作用小、不易产生耐药性、稳定持久的生物疗法,选择以肠杆菌作为表达宿主,在其中转入具有酪氨酸解氨酶的载体,从而使构建得到的工程菌能够高表达酪氨酸解氨酶,该酶可将胃肠内摄入的食源性酪氨酸降解。为了进一步满足有效治疗酪氨酸血症的目的,通过增添蛋白标签的方法来提高酪氨酸解氨酶的活性。且经验证,本发明的工程菌具有较强的对胃肠道消化***的抗性及耐受能力,能够制备成菌剂或与其他辅料配合后应用于降解体内酪氨酸,实现治疗酪氨酸血症的目的。
附图说明
图1为纯化后的蛋白活性检测结果;
图2为不同来源酪氨酸解氨酶导入大肠杆菌后的全菌体活性检测结果;
图3为表达元件优化后pGEX-4T-2质粒结构示意图;
图4为融合表达不同蛋白标签后对全菌体降解活性的影响;
图5为肠液耐受性实验结果;
图6为胃液耐受性实验结果。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明,以使本领域的技术人员可以更好地理解本发明并能予以实施,但所举实施例不作为对本发明的限定。
下述实施例所涉及的检测方法:
(1)酪氨酸检测方法:酪氨酸作为反应底物,通过检测酪氨酸在反应体系中的降低情况,建立并优化了液相色谱检测方法,检测采用岛津高效液相色谱LC-2050C,检测条件参数如下:
Tyr液相检测方法
色谱柱:SHIMADZU C18色谱柱(250mm×4.6mm,5μm);
流动相:1%乙酸和乙腈;
柱温:40℃;
流速:0.8mL/min
梯度洗脱:
0-8min,1.5%乙酸—乙腈(95:5)渐变至1.5%乙酸—乙腈(0:100);
8-13min,保持1.5%乙酸—乙腈(0:100)
13-14min,1.5%乙酸—乙腈(0:100)渐变至1.5%乙酸—乙腈(95:5);
14-23min,保持1.5%乙酸—乙腈(95:5)
检测:紫外,波长280nm
进样量:10μL
(2)全菌体酪氨酸解氨酶的降解活性检测条件为:反应时间3h、37℃水浴、底物1mMTyr、菌体量1*1010。
下述实施例所涉及的材料如下:
SOC液体培养基:2%胰蛋白胨、0.5%酵母提取物、0.05% NaCl、2.5mM KCl、10mMMgCl2、10mM MgSO4、20mM D-葡萄糖,调节pH至7.5。
模拟胃液和肠液:均购自南京源之信生物技术有限公司,于-20℃冰箱中保存。
下述实施例中所涉及的序列如下:
编码酪氨酸解氨酶FjTAL的氨基酸序列如SEQ ID NO.1所示;
编码酪氨酸解氨酶SeSam8的氨基酸序列如SEQ ID NO.2所示;
编码酪氨酸解氨酶TcPAL的氨基酸序列如SEQ ID NO.3所示;
编码酪氨酸解氨酶PcXAL的氨基酸序列如SEQ ID NO.4所示;
编码酪氨酸解氨酶RtXAL的氨基酸序列如SEQ ID NO.5所示;
编码酪氨酸解氨酶RmXAL的氨基酸序列如SEQ ID NO.6所示;
编码酪氨酸解氨酶FjTAL的核苷酸序列如SEQ ID NO.7所示;
编码SUMO标签的核苷酸序列如SEQ ID NO.8所示;
编码GST标签的核苷酸序列如SEQ ID NO.9所示;
编码GroE标签的核苷酸序列如SEQ ID NO.10所示。
实施例1酪氨酸解氨酶的筛选及其降解酪氨酸能力的研究
利用基于数据库的宏筛选技术,对NCBI、PDB、Uniprot等数据库中不同来源的酪氨酸解氨酶进行虚拟筛选及功能预测,将全基因合成得分较高的酪氨酸解氨酶进行体外表达及活性验证,选出纯化后酶活力较高的酶,结果见图1。对筛选出的6种酶进行重组表达:以质粒pGEX-4T-2作为表达载体,以其核苷酸序列设计引物进行扩增,目的基因经过PCR扩增后通过酶切连接方法连接至表达载体,验证序列无误后进行发酵检测。
具体如下:质粒pGEX-4T-2酶切后按照诺唯赞胶回收盒(DC301-01)说明进行纯化回收。使用引物FjTAL-F和FjTAL-R以及pUC57-FjTAL作为模板,PCR扩增得到FjTAL片段,胶回收纯化(DC301-01);使用引物SeSam8-F和SeSam8-R以及pUC57-SeSam8作为模板,PCR扩增得到SeSam8片段,胶回收纯化(DC301-01);使用引物PcXAL-F和PcXAL-R以及pUC57-PcXAL作为模板,PCR扩增得到PcXAL片段,胶回收纯化(DC301-01);使用引物TcPAL-F和TcPAL-R以及pUC57-TcPAL作为模板,PCR扩增得到TcPAL片段,胶回收纯化(DC301-01);使用引物RtXAL-F和RtXAL-R以及pUC57-RtXAL作为模板,PCR扩增得到RtXAL片段,胶回收纯化(DC301-01);使用引物RmXAL-F和RmXAL-R以及pUC57-RmXAL作为模板,PCR扩增得到RmXAL片段,胶回收纯化(DC301-01);将纯化后的上述片段使用T4 DNA连接酶进行连接,电转化大肠杆菌EcN1917,经过测序验证得到含有正确的阳性克隆:pGEX-4T-2-FjTAL、pGEX-4T-2-SeSam8、pGEX-4T-2-TcPAL、pGEX-4T-2-PcXAL、pGEX-4T-2-RtXAL、pGEX-4T-2-RmXAL;挑取阳性克隆单克隆制备种子液,经发酵、诱导、测定酪氨酸解氨酶活性,结果如图2所示。
根据图1-2可知,纯化后的蛋白均具有较高Tyr降解活性,但是由于蛋白性质的差异,导致其在菌体内表达量、溶解度、活性存在较大差异,因此相同Cfu条件下全菌体活性也存在较大差异。图2中显示通过对筛选出的6种酪氨酸解氨酶进行全菌体活性检测,约氏黄杆菌(Flavobacterium johnsoniae)编码基因(命名为FjTAL,SEQ ID NO.7)在肠杆菌中表达所得到的酶的活性较高,可以高效降解酪氨酸。
表1扩增引物
名称 | 序列 | 酶切位点 |
FjTAL-F | CGGAATTCATGAACACCATCAACGAATACCT | EcoRI |
FjTAL-R | CCCTCGAGGTTGTTAATCAGGTGGTCTTTAACT | XhoI |
SeSam-F | CGGAATTCATGACCCAGGTGGTTGAACG | EcoRI |
SeSam-R | CCCTCGAGACCGAAATCTTTACCGTCAGCT | XhoI |
TcPAL-F | CGGAATTCATGTTCATTGAAACCAACGTGGCT | EcoRI |
TcPAL-R | CCCTCGAGACGACCGTCACGCATCG | XhoI |
PcXAL-F | CGGAATTCATGCCGAGCCGTATCGACTA | EcoRI |
PcXAL-R | CCCTCGAGCGCTTTGATGGATTTAACCAGC | XhoI |
RmXAL-F | CGGAATTCATGGCACCGAGCGTTGATAG | EcoRI |
RmXAL-R | CCCTCGAGCGCCATCATTTTAACCAGCACC | XhoI |
RtXAL-F | CGGAATTCATGGCTCCGAGCCTGGATTC | EcoRI |
RtXAL-R | CCCTCGAGCGCCAGCATTTTCAGCAGAA | XhoI |
实施例2表达元件优化
根据实施例1的结果,以FjTAL为主要研究对象。为进一步提高工程菌株活性,在FjTAL片段N端融合不同的蛋白标签(质粒构建示意图见图3),分别通过(G4S)*3linker连接,将融合表达SUMO标签、GST标签、GroE标签构建得到的工程菌株分别命名为FjTAL-2、FjTAL-3、FjTAL-4。结果见图4。其中,未添加蛋白标签的菌株FjTAL降解率为50%;添加SUMO标签后,FjTAL-2降解率为88.5%,较FjTAL提高77%;添加GST标签后,FjTAL-3降解率为59%,较FjTAL提高18%;添加GroE标签后,FjTAL-4降解率为69%,较FjTAL提高38%。综上所述,FjTAL-2、FjTAL-3、FjTAL-4具有优异的酪氨酸降解活性,可作为候选菌株进行进一步研究。
实施例3菌株的人工胃肠液抗性实验
1、材料准备:
(1)模拟胃液和肠液:将模拟胃液和肠液于4℃冰箱解冻。
(2)菌液制备:
将实施例2的菌株FjTAL、FjTAL-2、FjTAL-3、FjTAL-4,接种于SOC液体培养基中,当菌体生长到OD=0.6时加入IPTG,过夜诱导蛋白表达;次日,离心收集菌体,PBS洗涤3次,加保护剂重悬用于胃肠液耐受性实验。
2、胃肠液耐受性实验操作
吸取人工胃液/人工肠液接种于加有保护剂的菌体重悬液中,反应30min、60min、90min、120min、150min、180min后,利用HPLC的方法测定酪氨酸降解率。
结果见图5-6,其中,图5表示1*1010CFU菌体(含保护剂)与肠液(pH=6.8)1:1混合后的酪氨酸降解率,图6表示1*1010CFU菌体(含保护剂)与胃液(pH=2)1:1混合后的酪氨酸降解率。可见,工程菌FjTAL-4在人工胃肠液的耐受实验中表现更为优异,在该条件下的降解率相比于实施例2有进一步提升,说明其更适合在体内进行酪氨酸的降解,从而起到治疗或缓解酪氨酸血症的目的。
显然,上述实施例仅仅是为清楚地说明所作的举例,并非对实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
Claims (10)
1.一种表达酪氨酸解氨酶的重组大肠杆菌,其特征在于:所述重组大肠杆菌异源表达了氨基酸序列如SEQ ID NO.1所示的酪氨酸解氨酶FjTAL,并在所述酪氨酸解氨酶FjTAL的N端融合表达了蛋白标签。
2.根据权利要求1所述的重组大肠杆菌,其特征在于:所述蛋白标签选自SUMO标签、GST标签或GroE标签。
3.根据权利要求2所述的重组大肠杆菌,其特征在于:所述SUMO标签的核苷酸序列如SEQ ID NO.8所示;所述GST标签的核苷酸序列如SEQ ID NO.9所示;所述GroE标签的核苷酸序列如SEQ ID NO.10所示。
4.根据权利要求1所述的重组大肠杆菌,其特征在于:所述酪氨酸解氨酶FjTAL编码基因的核苷酸序列如SEQ ID NO.7所示。
5.根据权利要求1所述的重组大肠杆菌,其特征在于:以Escherichia coliNissle1917为出发菌株。
6.根据权利要求1所述的重组大肠杆菌,其特征在于:以pGEX-4T-2为表达载体。
7.权利要求1-6任一项所述的重组大肠杆菌的构建方法,其特征在于,包括以下步骤:
S1、将蛋白标签融合至酪氨酸解氨酶FjTAL的N端,并连接至表达载体上,得到重组质粒;
S2、将S1得到的重组质粒导入大肠杆菌宿主中,得到所述重组大肠杆菌。
8.权利要求1-6任一项所述的重组大肠杆菌在降解酪氨酸中的应用。
9.权利要求1-6任一项所述的重组大肠杆菌在制备酪氨酸血症预防或治疗药物中的应用。
10.一种可降解酪氨酸的微生物菌剂,其特征在于:含有权利要求1-6任一项所述重组大肠杆菌。
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