煙草節(jié)桿菌02181肌酸酶基因的克隆與表達(dá)研究
發(fā)布時間：2018-07-13 08:08
【摘要】： 血清和尿液中肌酐測定是臨床評價腎小球?yàn)V過功能的重要指標(biāo),因而是臨床上開展的重要生化項(xiàng)目之一。肌酐測定主要有化學(xué)法和酶法,這兩種方法都是基于比色法�；瘜W(xué)法主要源于Jeffe反應(yīng)法,其優(yōu)點(diǎn)是成本低,操作簡單,易于基層單位開展,現(xiàn)在仍有很多基層醫(yī)院在使用,但它的缺點(diǎn)也是明顯的,即易受到樣品中非特異性物質(zhì)的干擾,也就是所謂的“假肌酐”。酶法則克服了上述缺點(diǎn),無論是靈敏度還是特異性都有著化學(xué)法不可比擬的優(yōu)勢,但酶法測定肌酐也有自身的缺點(diǎn)。酶法所用的工具酶主要有三種(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種,主要來源于微生物,但在我國目前尚不能自主生產(chǎn)。臨床上肌酐酶學(xué)檢測,主要使用進(jìn)口原裝試劑盒或者國內(nèi)生物技術(shù)公司用進(jìn)口工具酶組裝的試劑盒,成本較化學(xué)法高出很多。國際臨床化學(xué)聯(lián)盟(International Federation ofClinical Chemists,IFCC)和我國臨床檢驗(yàn)中心(National Center for Clinical Laboratory,NCCL)推薦的肌酐測定方法是肌酐酶偶聯(lián)肌氨酸氧化酶法,在這一大趨勢下,開展具有完全自主知識產(chǎn)權(quán)的肌酐測定工具酶的研究具有特別重要的意義和良好的市場應(yīng)用前景。 主要的研究內(nèi)容和結(jié)果: 1.肌酸酶基因的克隆 1.1肌酸酶基因部分序列的克隆 查詢GenBank上已發(fā)表的全部肌酸酶基因序列,從中選出7個不同種屬來源的序列,將這7個序列遞交到Block Maker服務(wù)器上進(jìn)行塊狀比對,得到9個無間隙的保守區(qū),再通過CODEHOP服務(wù)器運(yùn)算,設(shè)計(jì)簡并引物,以提取的煙草節(jié)桿菌02181基因組DNA為模板做簡并PCR,得到一414 bp的片段,經(jīng)在NCBI上BLASTx,與多種不同來源的肌酸酶有著高度的同源性,證實(shí)該序列為肌酸酶基因的部分序列。 1.2肌酸酶基因全長序列的克隆 根據(jù)已有結(jié)果,按照基因組步移技術(shù)要求設(shè)計(jì)該肌酸酶基因部分序列上、下游基因特異性引物GSP1、GSP2,利用這一技術(shù)分別克隆出已知序列的上、下游序列,利用BLASTx、Vector NTI suit 8、primer premier 5.0等工具軟件拼接出肌酸酶基因的全長序列,該基因共有1254 bp(含有終止密碼子),為一完整的開放讀碼框架(ORF),編碼417個氨基酸,理論分子量為46377 Da。在NCBI上BLASTx結(jié)果顯示,所得肌酸酶基因與多種不同來源的肌酸酶基因高度同源,其中同源性最高的為Arthrobacter sp.FB24來源的肌酸酶,其氨基酸序列的一致性為79%(332/417),同源性達(dá)到了87%(365/417),證明我們得到是一種全新的肌酸酶基因。在此基礎(chǔ)之上,以該全長序列為模板,設(shè)計(jì)含有酶切位點(diǎn)及相應(yīng)保護(hù)堿基的引物,其中,上游引物包含Eco RⅠ及EK酶(小腸激酶)的酶切位點(diǎn),下游引物包含SalⅠ的酶切位點(diǎn),以煙草節(jié)桿菌02181基因組DNA為模板,利用pfu酶克隆出含有酶切位點(diǎn)的肌酸酶基因,該基因全長1288 bp。經(jīng)測序表明,所得序列與預(yù)期完全一致,證明我們克隆煙草節(jié)桿菌02181肌酸酶基因取得成功。 2.肌酸酶在大腸桿菌BL21(DE3)中的表達(dá)與純化 用Eco RⅠ及SalⅠ分別雙酶切PCR產(chǎn)物及pET42a質(zhì)粒,經(jīng)瓊脂糖凝膠電泳并回收純化后利用T4連接酶將該肌酸酶基因連入質(zhì)粒pET42a,然后轉(zhuǎn)化入大腸桿菌DH5α,酶切及測序以確認(rèn)其正確性。將該重組質(zhì)粒轉(zhuǎn)化入大腸桿菌BL21(DE3)表達(dá),經(jīng)SDS-PAGE電泳,得到一種分子量約67 kDa的蛋白(帶有GST標(biāo)簽,其分子量約為30 kDa),與預(yù)期完全相符。純化過程中,我們利用EK酶將GST標(biāo)簽切割下來,經(jīng)過第二輪GST純化后,可得到純度較高的不帶有任何“外來”氨基酸的肌酸酶,經(jīng)SDS-PAGE電泳,確定其單體分子量為46.4 kDa。經(jīng)酶活性測定,每克濕菌可產(chǎn)生156.8 U的肌酸酶,與野生型煙草節(jié)桿菌每克濕菌產(chǎn)生10.7 U肌酸酶相比,產(chǎn)量提高了14.7倍,并且建立了重組肌酸酶的純化工藝,純化后的肌酸酶的比活性較純化前提高了24.7倍。經(jīng)純化得到的肌酸酶在37℃時最適作用pH值為6.0,37℃時也很穩(wěn)定,于40℃溫育30 min后活性即開始下降,但在此溫度下孵育60 min后酶活性再無明顯降低,而在50℃下溫育30 min后則活性完全喪失,從這些指標(biāo)來看,已能滿足臨床應(yīng)用的需要,為實(shí)現(xiàn)肌酐測定試劑盒的國產(chǎn)化奠定了良好的基礎(chǔ)。
[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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R346
本文編號：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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R346
【參考文獻(xiàn)】
相關(guān)期刊論文 前10條
1 楊麗君,郁衛(wèi)東,梁蓉,尚美,郭靜竹;基因組簡并寡核苷酸引物聚合酶鏈反應(yīng)的影響因素[J];北京大學(xué)學(xué)報(醫(yī)學(xué)版);2005年04期
2 段金秀;郭麗;曹致中;張向東;王小行;郭蜀光;;杜氏鹽藻CDPK基因的克隆及其序列的分析[J];草業(yè)科學(xué);2006年09期
3 羅侃;崔有宏;曾志南;徐東;賀燕鴻;;煙草節(jié)桿菌02181肌酸酶的純化及理化特性[J];甘肅科學(xué)學(xué)報;2006年01期
4 黃菁,王少麗,喬傳令;程序化設(shè)計(jì)簡并引物與克隆小菜蛾酯酶基因[J];昆蟲知識;2002年06期
5 丁兆軍,王臺,種康;水稻減數(shù)分裂相關(guān)基因OsDMC1的克隆[J];科學(xué)通報;2001年10期
6 鄭仲承;寡核苷酸的優(yōu)化設(shè)計(jì)[J];生命的化學(xué);2001年03期
7 崔郁敏;李貴生;廖婉琴;梁旭方;;斑鱧肝臟解偶聯(lián)蛋白2cDNA核心片斷的克隆及分析[J];生態(tài)科學(xué);2006年02期
8 羅侃,崔有宏,鄭強(qiáng),支東學(xué),王緒明;臨床生化檢驗(yàn)工具酶的基因克隆[J];現(xiàn)代檢驗(yàn)醫(yī)學(xué)雜志;2003年01期
9 崔有宏,羅侃,鄭強(qiáng),王緒明;肌酐測定的酶學(xué)方法[J];西北國防醫(yī)學(xué)雜志;2003年02期
10 劉濤;李曉賢;徐平珍;周浙昆;胡運(yùn)乾;;板栗MADS-box蛋白基因(CmMADS3)的克隆和表達(dá)分析[J];云南植物研究;2006年03期
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