烟草节杆菌02181肌酸酶基因的克隆与表达研究
发布时间：2018-07-13 08:08
【摘要】： 血清和尿液中肌酐测定是临床评价肾小球滤过功能的重要指标,因而是临床上开展的重要生化项目之一。肌酐测定主要有化学法和酶法,这两种方法都是基于比色法。化学法主要源于Jeffe反应法,其优点是成本低,操作简单,易于基层单位开展,现在仍有很多基层医院在使用,但它的缺点也是明显的,即易受到样品中非特异性物质的干扰,也就是所谓的“假肌酐”。酶法则克服了上述缺点,无论是灵敏度还是特异性都有着化学法不可比拟的优势,但酶法测定肌酐也有自身的缺点。酶法所用的工具酶主要有三种(肌酐酶,EC 3.5.2.10;肌酸酶,EC 3.5.3.3;肌氨酸氧化酶,EC 1.5.3.1),肌酸酶是其中非常重要的一种,主要来源于微生物,但在我国目前尚不能自主生产。临床上肌酐酶学检测,主要使用进口原装试剂盒或者国内生物技术公司用进口工具酶组装的试剂盒,成本较化学法高出很多。国际临床化学联盟(International Federation ofClinical Chemists,IFCC)和我国临床检验中心(National Center for Clinical Laboratory,NCCL)推荐的肌酐测定方法是肌酐酶偶联肌氨酸氧化酶法,在这一大趋势下,开展具有完全自主知识产权的肌酐测定工具酶的研究具有特别重要的意义和良好的市场应用前景。 主要的研究内容和结果: 1.肌酸酶基因的克隆 1.1肌酸酶基因部分序列的克隆 查询GenBank上已发表的全部肌酸酶基因序列,从中选出7个不同种属来源的序列,将这7个序列递交到Block Maker服务器上进行块状比对,得到9个无间隙的保守区,再通过CODEHOP服务器运算,设计简并引物,以提取的烟草节杆菌02181基因组DNA为模板做简并PCR,得到一414 bp的片段,经在NCBI上BLASTx,与多种不同来源的肌酸酶有着高度的同源性,证实该序列为肌酸酶基因的部分序列。 1.2肌酸酶基因全长序列的克隆 根据已有结果,按照基因组步移技术要求设计该肌酸酶基因部分序列上、下游基因特异性引物GSP1、GSP2,利用这一技术分别克隆出已知序列的上、下游序列,利用BLASTx、Vector NTI suit 8、primer premier 5.0等工具软件拼接出肌酸酶基因的全长序列,该基因共有1254 bp(含有终止密码子),为一完整的开放读码框架(ORF),编码417个氨基酸,理论分子量为46377 Da。在NCBI上BLASTx结果显示,所得肌酸酶基因与多种不同来源的肌酸酶基因高度同源,其中同源性最高的为Arthrobacter sp.FB24来源的肌酸酶,其氨基酸序列的一致性为79%(332/417),同源性达到了87%(365/417),证明我们得到是一种全新的肌酸酶基因。在此基础之上,以该全长序列为模板,设计含有酶切位点及相应保护碱基的引物,其中,上游引物包含Eco RⅠ及EK酶(小肠激酶)的酶切位点,下游引物包含SalⅠ的酶切位点,以烟草节杆菌02181基因组DNA为模板,利用pfu酶克隆出含有酶切位点的肌酸酶基因,该基因全长1288 bp。经测序表明,所得序列与预期完全一致,证明我们克隆烟草节杆菌02181肌酸酶基因取得成功。 2.肌酸酶在大肠杆菌BL21(DE3)中的表达与纯化 用Eco RⅠ及SalⅠ分别双酶切PCR产物及pET42a质粒,经琼脂糖凝胶电泳并回收纯化后利用T4连接酶将该肌酸酶基因连入质粒pET42a,然后转化入大肠杆菌DH5α,酶切及测序以确认其正确性。将该重组质粒转化入大肠杆菌BL21(DE3)表达,经SDS-PAGE电泳,得到一种分子量约67 kDa的蛋白(带有GST标签,其分子量约为30 kDa),与预期完全相符。纯化过程中,我们利用EK酶将GST标签切割下来,经过第二轮GST纯化后,可得到纯度较高的不带有任何“外来”氨基酸的肌酸酶,经SDS-PAGE电泳,确定其单体分子量为46.4 kDa。经酶活性测定,每克湿菌可产生156.8 U的肌酸酶,与野生型烟草节杆菌每克湿菌产生10.7 U肌酸酶相比,产量提高了14.7倍,并且建立了重组肌酸酶的纯化工艺,纯化后的肌酸酶的比活性较纯化前提高了24.7倍。经纯化得到的肌酸酶在37℃时最适作用pH值为6.0,37℃时也很稳定,于40℃温育30 min后活性即开始下降,但在此温度下孵育60 min后酶活性再无明显降低,而在50℃下温育30 min后则活性完全丧失,从这些指标来看,已能满足临床应用的需要,为实现肌酐测定试剂盒的国产化奠定了良好的基础。
[Abstract]:The determination of creatinine in serum and urine is an important indicator of clinical evaluation of glomerular filtration function, so it is one of the most important biochemical items in clinical. The determination of creatinine is mainly chemical and enzyme methods. These two methods are based on colorimetric method. Chemical method is mainly derived from Jeffe reaction, which has the advantages of low cost, simple operation and easy to be used. There are still a lot of grass-roots hospitals being used, but its shortcomings are also obvious, that is, it is easy to be disturbed by the non specific substances in the sample, that is, the so-called "false creatinine". The enzyme rule overcomes the above shortcomings, both sensitivity and specificity have the incomparable advantages of chemical method, but the enzymatic method for the determination of creatinine also has self - determination. There are three main enzymes used in the enzyme method (creatinase, EC 3.5.2.10; Creatine enzyme, EC 3.5.3.3; Creatine oxidase, EC 1.5.3.1). The creatine enzyme is a very important one, mainly from microbes, but it is not produced in our country at present. The domestic Biotech Corp uses a kit of imported tool enzymes, which is much higher than the chemical method. The creatinine determination method of the International Federation ofClinical Chemists (IFCC) and the clinical laboratory center of our country (National Center for Clinical Laboratory, NCCL) is the creatinine coupled creatinine coupled with creatinine In this trend, it is of special significance and good market prospect to carry out the research of creatinine enzyme with fully autonomous intellectual property right under this trend.
The main research contents and results are as follows:
Cloning of 1. creatine enzyme gene
Cloning of the partial sequence of 1.1 creatine enzyme gene
The sequence of all creatine enzyme genes published on GenBank was searched, and 7 sequences of different species were selected. The 7 sequences were submitted to the Block Maker server for massive comparison, and 9 gap conservative regions were obtained. Then the CODEHOP server was used to design the degenerate primers to extract the genomic DNA of Bacillus Arthrobacter 02181. The template is degenerate and PCR, and a fragment of a 414 BP is obtained. By BLASTx on NCBI, it has a high homology with a variety of different sources of creatine enzyme, which confirms that this sequence is a partial sequence of the creatine enzyme gene.
Cloning of the full length sequence of 1.2 creatine enzyme gene
According to the existing results, the specific sequence of the creatine enzyme gene was designed according to the genomic step technique, the downstream gene specific primers GSP1, GSP2, using this technique to clone the upper and downstream sequences of the known sequences, and to splice the full-length sequence of the creatine enzyme gene by using BLASTx, Vector NTI suit 8, primer premier 5 and other tools. The gene has a total of 1254 BP (containing terminating codon), a complete open code framework (ORF), encoding 417 amino acids, and the theoretical molecular weight of 46377 Da. on NCBI BLASTx results showed that the creatine enzyme gene was highly homologous to a variety of different sources of the creatine enzyme gene, of which the highest homology was the creatine source of Arthrobacter sp.FB24. The homology of the amino acid sequence is 79% (332/417) and the homology is 87% (365/417). It is proved that we have been a new creatine enzyme gene. Based on this, the full length sequence is used as the template to design the primers containing the enzyme cut site and the corresponding protection base. The upstream primers include the enzyme digestion of the Eco R I and the EK enzyme (small intestine kinase). The loci, the downstream primers contained the enzyme cutting site of Sal I, and cloned the creatine enzyme gene containing the enzyme cut site using the PFU enzyme of DNA as the template. The whole length of the gene was sequenced. The sequence showed that the sequence was exactly the same as expected. It proved that our clone of bacilli bacilli 02181 creatine gene was successful.
Expression and purification of creatinase 2. in Escherichia coli BL21 (DE3)
Eco R I and Sal I were used to cut PCR products and pET42a plasmids respectively. After agarose gel electrophoresis and recovery, the creatine enzyme gene was linked into plasmid pET42a, and then transformed into Escherichia coli DH5 a, enzyme digestion and sequencing to confirm its correctness. The recombinant plasmid was transformed into Escherichia coli BL21 (DE3) expression, and SDS-PAGE electricity was used. In swimming, a protein with a molecular weight of about 67 kDa (with a GST tag with a molecular weight of about 30 kDa) is completely consistent with expectation. In the purification process, we use EK enzyme to cut the GST label and after second rounds of GST purification, we can obtain a high purity creatine enzyme without any "outside" amino acid, and determine its monomer by SDS-PAGE electrophoresis. The body molecular weight of 46.4 kDa. was measured by enzyme activity. The myocreatase was 156.8 U per gram of humid bacteria. Compared with 10.7 U myocreatase from the wild type bacilli, the yield was increased by 14.7 times, and the purification process of the recombinant creatine enzyme was established. The specific activity of the purified creatine enzyme was 24.7 times higher than that before the purification. The optimum pH value of creatine enzyme at 37 C was also stable at 6.0,37 C. The activity began to decrease after temperature breeding 30 min at 40 C, but after incubating 60 min at this temperature, the activity of enzyme did not decrease obviously, and the activity was completely lost after 30 min at 50 temperature. From these indexes, it could meet the needs of clinical application and achieve creatinine measurement. The localization of the reagent kit has laid a good foundation.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R346
本文编号：2118731
[Abstract]:The determination of creatinine in serum and urine is an important indicator of clinical evaluation of glomerular filtration function, so it is one of the most important biochemical items in clinical. The determination of creatinine is mainly chemical and enzyme methods. These two methods are based on colorimetric method. Chemical method is mainly derived from Jeffe reaction, which has the advantages of low cost, simple operation and easy to be used. There are still a lot of grass-roots hospitals being used, but its shortcomings are also obvious, that is, it is easy to be disturbed by the non specific substances in the sample, that is, the so-called "false creatinine". The enzyme rule overcomes the above shortcomings, both sensitivity and specificity have the incomparable advantages of chemical method, but the enzymatic method for the determination of creatinine also has self - determination. There are three main enzymes used in the enzyme method (creatinase, EC 3.5.2.10; Creatine enzyme, EC 3.5.3.3; Creatine oxidase, EC 1.5.3.1). The creatine enzyme is a very important one, mainly from microbes, but it is not produced in our country at present. The domestic Biotech Corp uses a kit of imported tool enzymes, which is much higher than the chemical method. The creatinine determination method of the International Federation ofClinical Chemists (IFCC) and the clinical laboratory center of our country (National Center for Clinical Laboratory, NCCL) is the creatinine coupled creatinine coupled with creatinine In this trend, it is of special significance and good market prospect to carry out the research of creatinine enzyme with fully autonomous intellectual property right under this trend.
The main research contents and results are as follows:
Cloning of 1. creatine enzyme gene
Cloning of the partial sequence of 1.1 creatine enzyme gene
The sequence of all creatine enzyme genes published on GenBank was searched, and 7 sequences of different species were selected. The 7 sequences were submitted to the Block Maker server for massive comparison, and 9 gap conservative regions were obtained. Then the CODEHOP server was used to design the degenerate primers to extract the genomic DNA of Bacillus Arthrobacter 02181. The template is degenerate and PCR, and a fragment of a 414 BP is obtained. By BLASTx on NCBI, it has a high homology with a variety of different sources of creatine enzyme, which confirms that this sequence is a partial sequence of the creatine enzyme gene.
Cloning of the full length sequence of 1.2 creatine enzyme gene
According to the existing results, the specific sequence of the creatine enzyme gene was designed according to the genomic step technique, the downstream gene specific primers GSP1, GSP2, using this technique to clone the upper and downstream sequences of the known sequences, and to splice the full-length sequence of the creatine enzyme gene by using BLASTx, Vector NTI suit 8, primer premier 5 and other tools. The gene has a total of 1254 BP (containing terminating codon), a complete open code framework (ORF), encoding 417 amino acids, and the theoretical molecular weight of 46377 Da. on NCBI BLASTx results showed that the creatine enzyme gene was highly homologous to a variety of different sources of the creatine enzyme gene, of which the highest homology was the creatine source of Arthrobacter sp.FB24. The homology of the amino acid sequence is 79% (332/417) and the homology is 87% (365/417). It is proved that we have been a new creatine enzyme gene. Based on this, the full length sequence is used as the template to design the primers containing the enzyme cut site and the corresponding protection base. The upstream primers include the enzyme digestion of the Eco R I and the EK enzyme (small intestine kinase). The loci, the downstream primers contained the enzyme cutting site of Sal I, and cloned the creatine enzyme gene containing the enzyme cut site using the PFU enzyme of DNA as the template. The whole length of the gene was sequenced. The sequence showed that the sequence was exactly the same as expected. It proved that our clone of bacilli bacilli 02181 creatine gene was successful.
Expression and purification of creatinase 2. in Escherichia coli BL21 (DE3)
Eco R I and Sal I were used to cut PCR products and pET42a plasmids respectively. After agarose gel electrophoresis and recovery, the creatine enzyme gene was linked into plasmid pET42a, and then transformed into Escherichia coli DH5 a, enzyme digestion and sequencing to confirm its correctness. The recombinant plasmid was transformed into Escherichia coli BL21 (DE3) expression, and SDS-PAGE electricity was used. In swimming, a protein with a molecular weight of about 67 kDa (with a GST tag with a molecular weight of about 30 kDa) is completely consistent with expectation. In the purification process, we use EK enzyme to cut the GST label and after second rounds of GST purification, we can obtain a high purity creatine enzyme without any "outside" amino acid, and determine its monomer by SDS-PAGE electrophoresis. The body molecular weight of 46.4 kDa. was measured by enzyme activity. The myocreatase was 156.8 U per gram of humid bacteria. Compared with 10.7 U myocreatase from the wild type bacilli, the yield was increased by 14.7 times, and the purification process of the recombinant creatine enzyme was established. The specific activity of the purified creatine enzyme was 24.7 times higher than that before the purification. The optimum pH value of creatine enzyme at 37 C was also stable at 6.0,37 C. The activity began to decrease after temperature breeding 30 min at 40 C, but after incubating 60 min at this temperature, the activity of enzyme did not decrease obviously, and the activity was completely lost after 30 min at 50 temperature. From these indexes, it could meet the needs of clinical application and achieve creatinine measurement. The localization of the reagent kit has laid a good foundation.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R346
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