CN116240188A - 一种热稳定性提高的二氢蝶酸合成酶突变体的制备方法 - Google Patents
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Abstract
本发明涉及一种热稳定性提高的二氢蝶酸合成酶突变体的制备方法。所述制备方法包括获得受体蛋白‑DHPPP晶体复合物与SMM作用的氨基酸位点;对配体
Description
技术领域
本发明涉及结构生物学、生物信息学籍免疫学领域,具体涉及一种热稳定性提高的二氢蝶酸合成酶突变体的制备方法。
背景技术
免疫分析方法是目前磺胺类药物残留检测常用方法之一,其原理是基于抗原抗体的特异性结合,免疫分析方法具有高灵敏度、高特异性的特性,且具备高通量、检测快速、操作简便、适用于现场筛查等优势,已广泛应用于磺胺类药物残留筛查。
免疫分析方法的核心试剂是抗体,常规的抗体制备方法主要是免疫动物获得多克隆血清以及采用杂交瘤技术获得单克隆抗体,抗体的灵敏度虽然高,但获得抗体的周期长、过程复杂、费用高,且可识别所有磺胺类药物的抗体难以获得,因此制约了免疫分析方法在兽药残留检测领域的应用。
受体蛋白是一类药物分子真正意义上的广谱识别材料,制备简便,可均一识别一大类药物分子,是解决残留分析技术瓶颈问题的突破口之一。二氢蝶酸合成酶(Dihydropteroate Synthase,DHPS)是叶酸合成的催化酶之一,是磺胺类药物的受体蛋白,至少可识别28种磺胺类药物。目前已经有基于DHPS的磺胺类药物免疫分析方法,但是由于其热稳定性差,限制了其在实际样品中磺胺类药物残留的检测。如能获得热稳定性提高的DHPS突变体将有助于实际样品中磺胺类药物检测。
发明内容
基于现有技术中的缺陷,本发明的目的是提供一种热稳定性提高的二氢蝶酸合成酶突变体及其制备方法。
第一方面,本发明提供一种二氢蝶酸合成酶突变体MDHPS,所述二氢蝶酸合成酶突变体MDHPS,包括,野生型氨基酸序列第238位的ARG突变为TRP、第245位的GLU突变为CYS、第284位的HIS突变为TRP、第235位的SER突变为VAL和第251位的VAL突变为ILE。
具体地,本发明提供的二氢蝶酸合成酶突变体MDHPS的氨基酸序列如SEQ ID NO.2所示。
MSSKANHAKTVICGIINVTPDSFSDGGQFFALEQALQQARKLIAEGASMLDIGGESTRPGSSYVEIEEEIQRVVPVIKAIRKESDVLISIDTWKSQVAEAALAAGADLVNDITGLMGDEKMPHVVAEARAQVVIMFNPVMARPQHPSSLIFPHFGFGQAFTEEELADFETLPIEELMEAFFERALARAAEAGIAPENILLDPGIGFGLTKKENLLLLRDLDKLHQKGYPIFLGVVRKWFVINILCENGFEINPETELGFRNRDTASAHVTSIAARQGVEVVRVWDVASHRMAVEIASAIRLADEAENLDLKQYK。
第二方面,本发明提供上述二氢蝶酸合成酶突变体MDHPS的编码基因,所述编码基因的核苷酸序列如SEQ ID NO.1所示。
ATGAGCAGCAAAGCGAACCATGCAAAAACCGTAATTTGTGGTATTATTAACGTGACGCCGGATAGCTTTAGCGATGGTGGTCAGTTTTTTGCACTGGAACAGGCGCTGCAGCAGGCACGTAAACTGATCGCAGAAGGTGCATCCATGCTGGATATTGGTGGTGAAAGCACACGCCCTGGTTCAAGCTATGTTGAAATTGAAGAAGAAATCCAGCGTGTTGTTCCGGTTATTAAAGCAATTCGTAAAGAAAGCGATGTTCTGATTTCTATTGATACATGGAAATCACAGGTTGCAGAAGCAGCACTGGCGGCAGGTGCAGATCTGGTTAATGATATTACCGGTCTGATGGGTGATGAAAAAATGCCGCATGTTGTTGCAGAAGCACGTGCACAGGTTGTTATTATGTTTAATCCGGTTATGGCACGTCCGCAGCATCCGAGCAGCCTGATTTTTCCGCATTTTGGTTTTGGTCAGGCATTTACCGAAGAAGAACTGGCAGATTTTGAAACCCTGCCGATTGAAGAACTGATGGAAGCATTTTTTGAACGTGCACTGGCACGTGCAGCAGAAGCAGGTATTGCACCGGAAAATATTCTGCTGGATCCGGGTATTGGTTTTGGTCTGACCAAAAAAGAAAATCTGCTGCTGCTGCGTGATCTGGATAAACTGCATCAGAAAGGTTATCCGATTTTTCTGGGTGTTGTTCGTAAATGGTTTGTTATTAATATTCTGTGTGAAAATGGTTTTGAAATTAATCCGGAAACCGAACTGGGTTTTCGTAATCGTGATACCGCAAGCGCACATGTTACCAGCATTGCAGCACGTCAGGGTGTTGAAGTTGTTCGTGTTTGGGATGTTGCAAGCCATCGTATGGCAGTTGAAATTGCAAGCGCAATTCGTCTGGCAGATGAAGCAGAAAATCTGGATCTGAAACAGTATAAA。
第三方面,本发明提供一种生物材料,所述生物材料可表达出上述的二氢蝶酸合成酶突变体MDHPS或所述生物材料含有上述的编码基因;所述生物材料为重组DNA、表达盒、转座子、质粒载体、噬菌体载体、病毒载体、工程菌或转基因细胞系。
根据本领域技术人员的理解,本发明请求保护,上述二氢蝶酸合成酶突变体MDHPS或上述的编码基因或上述的生物材料在磺胺类药物残留检测中的应用。
第四方面,本发明提供一种检测磺胺类药物残留的试剂或试剂盒,所述试剂或试剂盒,以上述二氢蝶酸合成酶突变体MDHPS作为磺胺类药物残留检测的受体蛋白。
第五方面,本发明提供一种检测磺胺类药物残留的方法,采用上述的二氢蝶酸合成酶突变体MDHPS作为磺胺类药物残留检测的免疫受体蛋白。
第六方面,本发明提供一种用于检测磺胺类药物残留的热稳定性受体蛋白的制备方法,包括:
(1)获得受体蛋白-DHPPP晶体复合物与SMM作用的氨基酸位点;
(3)获得受体蛋白热稳定性的关键氨基酸;
(4)通过软件模拟关键氨基酸突变前后的稳定力及亲和力确定突变氨基氨酸;
(5)基于所述突变氨基酸,构建热稳定性受体蛋白的氨基酸序列。
在本发明提供的制备方法中,所述热稳定性受体蛋白为二氢蝶酸合成酶突变体MDHPS。
具体地,本发明提供了一种利用计算机图形学等技术提高DHPS热稳定性的方法,包括以下步骤:
基于PDB结构信息数据库信息,将肺炎链球菌R6的DHPS-DHPPP晶体复合物(2VEG)空间结构信息导入Discovery Studio 2019软件中;
利用DHPS-DHPPP复合物与SMM进行分子对接,通过对接结果显示,DHPS-DHPPP复合物与磺胺药物SMM作用的关键氨基酸位于:ARG212、ARG214、ARG238、GLU221、HIS260、ILE217、LYS213、PRO229、SER211和VAL227;
选择对配体以内氨基酸分别进行热稳定性和亲和力的单点突变。本发明通过Discovery Studio 2019软件确定影响受体蛋白热稳定性的关键氨基酸,热稳定高的受体蛋白DHPS氨基酸对应位点为ARG238>TRP,GLU245>CYS,HIS284>TRP,SER235>VAL,VAL251>ILE。
因此,本发明提供的二氢蝶酸合成酶突变体MDHPS的氨基酸序列是由野生型二氢蝶酸合成酶氨基酸序列的第238位的ARG突变为TRP、第245位的GLU突变为CYS、第284位的HIS突变为TRP、第235位的SER突变为VAL和第251位的VAL突变为ILE获得的。
在本发明提供的制备方法中,当所述热稳定性受体蛋白为二氢蝶酸合成酶突变体MDHPS时,热稳定性受体蛋白的制备包括:编码所述二氢蝶酸合成酶突变体MDHPS的目的片段mdhps经重叠延伸PCR技术,由SEQ ID NO.3-24所示的核苷酸序列进行拼接获得,SEQ IDNO.3所示的核苷酸序列中插入NheI酶切位点,SEQ ID NO.24所示的核苷酸序列中插入XhoI酶切位点;
表达载体pET-28a进行NdeI和XhoI双酶切。将目的片段连接到表达载体上,得到重组表达载体pET-28a-mdhps,重组表达载体pET-28a-mdhps转化到大肠杆菌BL21(DE3)中,得到高效表达重组肺炎链球菌二氢叶酸还原酶的重组表达菌株;
在表达菌株培养体系的OD600为0.6-0.8时,向所述发酵体系中加入IPTG,16-28℃条件下诱导5-16h,获得二氢蝶酸合成酶突变体MDHPS。
本发明的有益效果在于:
本发明提供一种精准、高效、用于提高受体蛋白热稳定性的方法,本发明利用计算机图形学、结构生物学及免疫学原理,获得一种热稳定提高的二氢蝶酸合成酶突变体MDHPS。
本发明从理论上探讨影响受体蛋白热稳定性的关键因素,结合生物学实验,证实了本发明提供的方法适用于获得热稳定性高的受体蛋白,为受体蛋白热稳定性的改造提供理论依据。
附图说明
图1为本发明中肺炎链球菌R6的DHPS氨基酸序列。
图2为本发明中DHPS的空间模型图。
图3为本发明中肺炎链球菌R6的晶体结构中DHPS氨基酸序列图。
图4为本发明中不同氨基酸突变与SMM的对接图。
图5为本发明中DHPS-SMM的二维相互作用图。
图6为本发明中突变体DNA片段mdhps的PCR验证图。
图7为本发明中突变体MDHPS的SDS-PAGE图。
图8为野生型DHPS和突变体MDHPS的热稳定性图。
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。
实施例1
本发明首先提供了一种利用计算机图形学等技术提高DHPS热稳定性的方法。其中,所述方法包括以下步骤:
A.从NCBI中GenBank上获取肺炎链球菌R6的DHPS氨基酸序列。经ExPASy数据库分析,DHPS共由314个氨基酸组成(图1),分子量约为35kDa,理论等电点为4.98,为酸性蛋白质,在中性环境中带负电荷。该蛋白的具体氨基酸组成见表1。其中负电性氨基酸(Asp+Glu)共47个,正电性氨基酸(Arg+Lys)共31个,摩尔消光系数为9970M-1cm-1,水中不稳定性系数为40.38,表明该酶属于不稳定蛋白。
表1二氢蝶酸合成酶的氨基酸组成
B.本发明基于PDB结构信息数据库信息,将肺炎链球菌R6的DHPS-DHPPP晶体复合物(2VEG)空间结构信息导入Discovery Studio2019软件中(图2),晶体氨基酸序列如图3。利用DHPS-DHPPP复合物与SMM进行分子对接。对接结果显示,其与SMM作用的关键氨基酸位于:ARG212、ARG214、ARG238、GLU221、HIS260、ILE217、LYS213、PRO229、SER211和VAL227。
C.选择对配体以内氨基酸分别进行热稳定性和亲和力的单点突变(图4)。本发明通过Discovery Studio 2019软件对DHPS-DHPPP赋予CHARMm力场,然后对氨基酸进行单点突变确定影响受体蛋白热稳定性的关键氨基酸:ARG212、ARG214、ARG238、GLU221、HIS260、ILE217、LYS213、PRO229、SER211和VAL227。
D.将10个氨基酸命名为Mutation,应用Discovery Studio 2019软件中DesignProtein模块的Calculate Mutation Energy(Stability)突变为其他19种氨基酸。
表2结果显示,当晶体结构中,ARG214>TRP(代表214位点由ARG突变为TRP)、GLU221>CYS、HIS260>TRP、SER211>VAL、VAL227>ILE时(此位点代表晶体结构中氨基酸位点,受体蛋白DHPS氨基酸对应位点为ARG238、GLU245、HIS284、SER235、VAL251)(图5),突变能均小于0.5,对应的Effect为稳定,说明这五个位点的突变均能使受体蛋白DHPS的热稳定性提高,亲和力保持不变或小幅提升,将该突变体命名为MDHPS。受体蛋白DHPS氨基酸对应位点为(ARG238>TRP,GLU245>CYS,HIS284>TRP,SER235>VAL,VAL251>ILE)。
实施例2突变体MDHPS的制备
本实施例提供突变体MDHPS的制备,具体步骤如下。
A.突变体肺炎链球菌R6的mdhps全基因设计:根据实施例1获得的MDHPS氨基酸序列,得到编码的基因序列SEQ ID NO.1。
B.以DHPS为研究对象,将要合成的双链基因分成22段,设计引物,在第1段与第22段插入酶切位点,其中,在SEQ ID NO.3中插入NheI酶切位点,SEQ ID NO.24中插入XhoI酶切位点,使每对相邻片段之间有15~16bp的交叉重叠,通过重叠延伸PCR技术将22个片段拼接,拼接后得到目的片段mdhps(表3)。
表3突变体mdhps引物设计
C.重组表达载体菌株的构建:将表达载体pET-28a进行NdeI和XhoI双酶切。将目的片段连接到表达载体上,得到重组表达载体pET-28a-mdhps。将重组表达载体pET-28a-mdhps转化到大肠杆菌BL21(DE3)中,筛选后得到高效表达重组肺炎链球菌二氢叶酸还原酶的重组表达菌株(图6)。
D.重组MDHPS的诱导表达:在表达菌株培养体系的OD600为0.6-0.8时,向所述发酵体系中加入IPTG,使IPTG在培养体系中的浓度为1mM。
E.重组MDHPS的纯化:将所述上清液进行镍柱亲和层析,收集洗脱液;所述镍柱亲和层析中,使用的洗脱缓冲液按照如下方法制备得到:将20mmol Tris、0.5mol NaCl、100mmol咪唑用水溶解,用HCl调pH值至7.9,再用水定容至1L,得到1L洗脱缓冲液。通过SDS-PAGE对纯化得到的MDHPS进行鉴定,结果见图7。
所述的重组DHPS的制备方法,其中,所述步骤C具体包括以下步骤:挑取单克隆重组表达菌株至含有30μg mL-1的卡那霉素的培养液中,经37℃,200rpm过夜培养后,将过夜菌液用液体培养基稀释100倍后37℃,200rpm培养3h。向培养液中加入终浓度为1M的IPTG,16-28℃条件下诱导5-16h,4℃条件下对培养液进行离心,收集菌体沉淀,用重悬缓冲液对菌体沉淀进行重悬后进行超声破碎,对悬液进行离心并收集上清液。
实施例3突变体MDHPS热稳定测试
以野生型DHPS和突变体MDHPS为研究对象,在40℃、45℃、50℃、55℃、60℃、65℃和70℃下分别热处理30min,测定蛋白与药物结合能力。以未热处理的最大吸收值为100%,不同温度处理的最大吸收值与未热处理最大吸收值的百分比作图,求出蛋白丧失50%结合能力时的温度,即蛋白的半失活温度(T50)。野生型和突变体在70℃热处理30min后基本都失活。如图8所示,野生型的T50值为52.3℃,突变体的T50值为55.5℃,比野生型的提高3.2℃,热稳定性有所提高,证实本方法可用于提高蛋白的热稳定性研究中。
虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
序列表
<110> 中国农业大学
<120> 一种热稳定性提高的二氢蝶酸合成酶突变体的制备方法
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Claims (10)
1.一种二氢蝶酸合成酶突变体MDHPS,其特征在于,所述二氢蝶酸合成酶突变体MDHPS包括5个点突变:野生型氨基酸序列第238位的ARG突变为TRP、第245位的GLU突变为CYS、第284位的HIS突变为TRP、第235位的SER突变为VAL和第251位的VAL突变为ILE。
2.根据权利要求1所述的二氢蝶酸合成酶突变体MDHPS,其特征在于,所述二氢蝶酸合成酶突变体MDHPS的氨基酸序列如SEQ ID NO.2所示。
3.权利要求1-2任一项所述二氢蝶酸合成酶突变体MDHPS的编码基因,所述编码基因的核苷酸序列如SEQ ID NO.1所示。
4.一种生物材料,其特征在于,所述生物材料可表达出权利要求1-2任一项所述的二氢蝶酸合成酶突变体MDHPS或所述生物材料含有权利要求3所述的编码基因;所述生物材料为重组DNA、表达盒、转座子、质粒载体、噬菌体载体、病毒载体、工程菌或转基因细胞系。
5.权利要求1-2任一项所述二氢蝶酸合成酶突变体MDHPS或权利要求3所述的编码基因或权利要求4所述的生物材料在磺胺类药物残留检测中的应用。
6.一种检测磺胺类药物残留的试剂或试剂盒,其特征在于,所述试剂或试剂盒,以权利要求1-2任一项所述二氢蝶酸合成酶突变体MDHPS作为磺胺类药物残留检测的受体蛋白。
7.一种检测磺胺类药物残留的方法,其特征在于,采用权利要求1-2任一项所述的二氢蝶酸合成酶突变体MDHPS作为磺胺类药物残留检测的免疫受体蛋白。
9.根据权利要求8所述的制备方法,其特征在于,所述热稳定性受体蛋白为二氢蝶酸合成酶突变体MDHPS。
10.根据权利要求9所述的制备方法,其特征在于,包括:编码所述二氢蝶酸合成酶突变体MDHPS的目的片段mdhps经重叠延伸PCR技术,由SEQ ID NO.3-24所示的核苷酸序列进行拼接获得,SEQ ID NO.3所示的核苷酸序列中插入NheI酶切位点,SEQ ID NO.24所示的核苷酸序列中插入XhoI酶切位点;
表达载体pET-28a进行NdeI和XhoI双酶切,将目的片段连接到表达载体上,得到重组表达载体pET-28a-mdhps,重组表达载体pET-28a-mdhps转化到大肠杆菌中,得到高效表达重组肺炎链球菌二氢叶酸还原酶的重组表达菌株;
在表达菌株培养体系的OD600为0.6-0.8时,向所述发酵体系中加入IPTG,16-28℃条件下诱导5-16h,获得二氢蝶酸合成酶突变体MDHPS。
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