WO2018133545A1 - 一株定点突变改造的基因工程精氨酸脱亚胺酶 - Google Patents

一株定点突变改造的基因工程精氨酸脱亚胺酶 Download PDF

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WO2018133545A1
WO2018133545A1 PCT/CN2017/113162 CN2017113162W WO2018133545A1 WO 2018133545 A1 WO2018133545 A1 WO 2018133545A1 CN 2017113162 W CN2017113162 W CN 2017113162W WO 2018133545 A1 WO2018133545 A1 WO 2018133545A1
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arginine deiminase
mutant
arginine
gene
enzyme
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张涛
江波
蒋航宇
沐万孟
缪铭
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江南大学
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Priority to US16/245,881 priority Critical patent/US10829755B2/en

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    • C12Y305/03—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amidines (3.5.3)
    • C12Y305/03006—Arginine deiminase (3.5.3.6)
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    • C12N2800/101—Plasmid DNA for bacteria

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  • the invention relates to a genetically engineered arginine deiminase modified by site-directed mutagenesis, belonging to the technical field of genetic engineering.
  • Arginine deiminase (EC 3.5.3.6) is referred to as ADI, which catalyzes the first reaction in the arginine metabolic pathway in microorganisms, that is, the hydrolysis of arginine and the formation of citrulline and ammonia. .
  • ADI Arginine deiminase
  • Arginine deiminase is widely available and is currently found in bacteria, archaea and some eukaryotic cells. At the same time, the properties of arginine deiminase from different sources are quite different.
  • arginine deiminase has a good performance in inhibiting arginine-deficient tumors, breast cancer, liver cancer cells, and as an alternative to L-asparaginase in the treatment of leukemia.
  • the ADI-PEG-20 developed by Phoenix USA has been conducting phase III human clinical trials of liver cancer worldwide. The results show that ADI-PEG-20 can extend the average life expectancy of patients by 76%.
  • arginine deiminase has not been widely used as a pharmaceutical enzyme due to problems such as low enzyme activity under physiological conditions, short half-life in vivo, and weak substrate affinity. Therefore, it is especially important to improve the enzymatic properties through molecular modification.
  • Sit-directed mutagenesis is one of the main means of molecular engineering, and refers to a technique of introducing a specific base pair at a designated site of a DNA fragment of interest, thereby changing the encoded amino acid sequence. Compared with other strategies to improve enzymatic properties, site-directed mutagenesis has the advantage of being more rapid, direct and cost-effective, and it is one of the means of genetic modification commonly used in laboratories.
  • the object of the present invention is to improve the enzyme activity and stability of the modified arginine deiminase under physiological pH 7.4 by molecular modification of arginine deiminase, and finally apply to The field of medicine.
  • a site-directed mutagenized arginine deiminase mutant called a genetically engineered arginine deiminase, arginine deacetylation by Enterococcus faecalis SK32.001
  • the amino acid sequence of the arginine deiminase mutant obtained by the site-directed mutagenesis is SEQ ID No. 1
  • the gene DNA of the arginine deiminase mutant of the site-directed mutation is Encoded by the nucleotide sequence of SEQ ID No. 2.
  • a recombinant plasmid comprising the DNA molecule.
  • a host cell comprising the DNA molecule or comprising the recombinant plasmid.
  • the arginine deiminase mutant, the arginine deiminase mutant plasmid containing the amino acid mutation point predicted by B-FITTER is transferred into the Escherichia coli BL21 (DE3) host, and the mutation is constructed. The sequence was confirmed to obtain a mutant Gly264Pro with an optimum pH and pH stability approaching physiological neutral pH 7.4, and the 264th glycine Gly was mutated to proline Pro.
  • the method for constructing the arginine deiminase mutant comprises the following steps:
  • the recombinant plasmid derived from the arginine deiminase gene carried by the E. coli host is used as a template, and the oligonucleotide sequence carrying the mutation site is used as a primer, and reverse PCR is carried out to amplify the full length of the mutant plasmid; Digestion with Dpn I restriction endonuclease; heat transfer of the PCR product treated with Dpn I restriction endonuclease to E. coli DH5 ⁇ , cultured on solid LB medium containing kanamycin resistance; Single colonies on solid LB medium were taken into liquid LB medium containing kanamycin resistance, and plasmids were extracted and sequenced. The plasmid with correct sequencing results was transformed into E. coli BL21 (DE3) competent state. In the cell, a mutant genetically engineered arginine deiminase was obtained.
  • arginine deiminase mutant The use of the arginine deiminase mutant, the arginine deiminase mutant is used for medicinal antitumor activity and related pharmacological activity studies.
  • the amino acid mutation is located inside the structure of the arginine deiminase protein, and the mutation can increase the hydrophobic interaction of the protein.
  • the amino acid was mutated by the substitution of glycine at position 264 of arginine deiminase to valine, and the resulting single mutant was named Gly264Pro.
  • the invention provides a method for constructing the arginine deiminase mutant, and the specific steps are as follows:
  • the mutated recombinant vector was transformed into E. coli BL21 (DE3) by heat shock, and the expression was induced, and the cells were collected. After sonication, the cells were separated and purified by Ni-NTA to obtain mutant arginine deimin. Enzyme.
  • the optimum pH and pH stability of the arginine deiminase mutant provided by the invention are shifted to the physiological neutral direction, and the optimum pH is increased from the original 5 to 5.5 to 5.5 to 7.5, and the pH stability range is from 5.5 to 6.5, offset to 6.5 to 7.5.
  • the problem that the wild type arginine deiminase has low catalytic activity and low stability under physiological pH 7.4 is solved, and the favorable conditions for the application of the enzyme in the medical field are created.
  • the gene arcA of the arginine deiminase used in the present invention is derived from a strain of Enterococcus faecalis which can produce citrulline, the strain number CCTCC NO: M 2011465, deposited in the China Center for Type Culture Collection, Address: Wuhan, China Wuhan University, named SK32.001 (Enterococcus faecalis SK32.001), has been published in Chinese patent CN 102433290 A.
  • Figure 1 Construction map of recombinant plasmid pET-28a-ADI.
  • Figure 2 Optimal pH profile of wild and mutant enzymes.
  • Figure 3 Plot stability changes of wild and mutant enzymes.
  • Enterococcus faecalis SK32.001 was cultured to the mid-exponential growth phase, and 2 mL of bacterial solution was centrifuged at 10000 r/min for 10 min to discard the supernatant. After lysozyme treatment for 30 min, the genomic DNA was extracted according to the kit instructions.
  • FADI-2 5'-CGCGGATCCATGAGTCATCCAATTAATGT-3' (containing BamH I restriction site),
  • RADI-2 5'-CCGCTCGAGTTAAAGATCTTCACGGT-3' (containing Xho I restriction site),
  • PCR amplification conditions denaturation at 95 ° C for 3 min, 30 cycles (95 ° C 30 s, 55 ° C 30 s, 72 ° C 210 s), and finally extended at 72 ° C for 2 min.
  • the PCR product and the vector pET-28a-c(+) were double-digested with BamH I and Xho I, and the digested products were separately recovered.
  • the solution was ligated with Solution I ligase at 16 ° C for 2 h for heat shock transformation.
  • the DH5 ⁇ cells after the transformants were grown, the single colonies were picked into LB medium, and the plasmid was extracted, and the recombinant plasmid pET-28a-ADI was verified by restriction enzyme digestion.
  • the plasmid was transformed into BL21 (DE3) cells to obtain BL21(DE3)/pET-28a-ADI engineered bacteria.
  • Primer design was performed based on the coding gene encoding arcA in Enterococcus faecalis SK23.001.
  • G264P-forward primer 5'-CTTGGCTTTTGATATCCCTGAACATCGTAAATTC-3'
  • G264P-reverse primer 5'-GATATCAAAAGCCAAGATATTTTTGAATCCTA-3'
  • the underlined portion represents the codon corresponding to the 264-glycine encoded by the mutant gene.
  • the PCR amplification system is:
  • Single colonies of BL21(DE3)/pET-28a-ADI and pET-28a-ADIG264P were picked up in LB medium containing 0.5mmol/L kanamycin, and cultured at 37°C, 200r/min for 12h, then transferred to In LB medium containing 0.5 mmol/L kanamycin, culture at 37 ° C, 200 r / min to OD 600 in the range of 0.5 ⁇ 0.7, add 1mmol / L IPTG at 28 ° C, 200r / min conditions induced 9h .
  • Ni 2+ chelated agarose resin column was pre-equilibrated with Binding Buffer; the crude enzyme solution was added and equilibrated with Binding Buffer and Washing Buffer respectively; the enzyme was eluted with Elution Buffer and recovered; the recovered enzyme solution was dialyzed in dialysis buffer After dialysis in the solution, it was stored in a refrigerator at 4 °C.
  • phosphate buffer (PB) 50mmol / L, pH 5.5;
  • 2BindingBuffer 50mmol / LPB, 500mmol / L NaCl, pH7.0;
  • 3Washing Buffer 50mmol/LPB, 500mmol/L NaCl, pH7.0, 50mmol/L imidazole;
  • 4Elution Buffer 50mmol/LPB, 500mmol/L NaCl, pH7.0, 500mmol/L imidazole;
  • Dialysis buffer 50 mmol/LPB, pH 5.5, 10 mmol/LEDTA.
  • Example 4 Determination of optimum pH and pH stability of wild and mutant enzymes
  • Enzyme activity assay method 0.5mL substrate L-arginine (concentration 10g / L), add 50mmol / L PB buffer 0.49mL at 45 ° C for 5min, then add 0.01mL enzyme solution at 45 ° C reaction After 10 min, the reaction was terminated by boiling for 10 min, and the reaction solution was centrifuged to remove the supernatant to determine the content of citrulline.
  • Enzyme activity definition The amount of enzyme required to produce 1 ⁇ mol of L -1 citrulline in 1 min is defined as one enzyme unit (U).
  • citrulline content high performance liquid chromatography: Angilent 1200; column: Hypersil ODS (5 ⁇ m, 4.0 mm ⁇ 250 mm); mobile phase A: water 2 L, sodium acetate trihydrate 13 g, triethylamine 0.4 mL, tetrahydrofuran 5 mL , pH 7.2 ⁇ 0.5; mobile phase B: 2L, sodium acetate trihydrate 15g, water/methanol/acetonitrile (volume ratio 1:2:2), pH 7.2 ⁇ 0.5; mobile phase A and B gradient elution, total Flow rate: 1.0 mL/min; column temperature: 40 ° C; injection amount: 10 ⁇ L; detector: ultraviolet detector; detection wavelength: 338 nm, emission wavelength 360 nm.
  • Optimum pH The wild enzyme or mutant enzyme pre-preserved in 50 mM PB buffer at pH 4.0-7.5 was placed in a 45 ° C water bath, and immediately after the reaction for 10 min, the reaction was terminated by boiling.
  • pH stability The same concentration of wild enzyme and mutant enzyme were pre-preserved in a buffer of pH 4.0-7.5 at 4 ° C for 12 h, and then the enzyme activity was measured at 45 ° C.

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Abstract

一株定点突变改造的基因工程精氨酸脱亚胺酶，属于基因工程技术领域。其氨基酸序列为SEQ ID No.1。所述定点突变的精氨酸脱亚胺酶氨基酸序列相对于天然的精氨酸脱亚胺酶氨基酸序列的第264位甘氨酸突变为脯氨酸。突变精氨酸脱亚胺酶与野生酶相比其有效pH作用范围得到一定的扩展，尤其在生理pH7.4下仍具有较好的酶活性。随着有效pH作用范围的拓宽，突变酶在pH 5.5～7.5条件下，仍然具有较高的稳定性。因此解决了精氨酸脱亚胺酶在临床上对肿瘤治疗的应用上生理条件下普遍酶活力低、体内半衰期短的问题，为利用该酶及其编码基因来用于临床治疗创造好的条件。

Description

一株定点突变改造的基因工程精氨酸脱亚胺酶 技术领域
本发明涉及一株定点突变改造的基因工程精氨酸脱亚胺酶，属于基因工程技术领域。
背景技术
精氨酸脱亚胺酶(Arginine deiminase，EC 3.5.3.6)简称ADI，它催化在微生物体内的精氨酸代谢途径中的第一个反应，即将精氨酸水解，并生成瓜氨酸和氨。精氨酸脱亚胺酶来源广泛，目前在细菌、古细菌及一些真核细胞中均有发现。同时，不同来源的精氨酸脱亚胺酶的性质有较大的差异。
目前，国内外研究精氨酸脱亚胺酶的学者主要将该酶应用于瓜氨酸的制备和疾病的治疗两大方面。其中医药领域上，精氨酸脱亚胺酶在抑制精氨酸缺陷性肿瘤、乳腺癌、肝癌细胞以及作为L-天冬酰胺酶的替代品治疗白血病方面具有良好的表现。美国凤凰公司研制的ADI-PEG-20已在全球进行肝癌的第Ⅲ期人体临床试验，结果表明ADI-PEG-20可使患者平均寿命延长76％。
迄今为止，由于作为药用酶受到在生理条件下酶活低、体内半衰期短、底物亲和性弱等问题的限制，精氨酸脱亚胺酶仍未大规模应用。因此通过分子改造以改善酶学性质尤为重要。
定点突变(sit-directed mutagenesis)是分子改造中的一种主要手段之一，是指在目的DNA片段的指定位点引入特定的碱基对，从而改变其编码的氨基酸序列的技术。与其他改善酶学性质的策略相比，定点突变具有更为迅速、直接和节约成本的优势，它是实验室常用的基因改造的手段之一。
发明内容
本发明的目的在于，通过对精氨酸脱亚胺酶进行分子改造，使改造后的精氨酸脱亚胺酶在生理pH7.4条件下的酶活和稳定性有所提高，最终应用于医药领域。
本发明的技术方案：一种定点突变改造的精氨酸脱亚胺酶突变体，称之为基因工程精氨酸脱亚胺酶，由粪肠球菌Enterococcus faecalis SK32.001的精氨酸脱亚胺酶基因出发，运用定点突变技术获得，所述的精氨酸脱亚胺酶突变体的氨基酸序列为SEQ ID No.1，定点突变的精氨酸脱亚胺酶突变体的基因DNA，是由SEQ ID No.2的核苷酸序列所编码。
一种重组质粒，其包含了所述的DNA分子。
一种宿主细胞，其含有所述的DNA分子，或含有所述的重组质粒。
所述的精氨酸脱亚胺酶突变体，将含有通过B-FITTER预测获得的氨基酸突变点的精氨酸脱亚胺酶突变质粒转入大肠杆菌Escherichia coli BL21(DE3)宿主中，构建突变体，进行序列验证确认，得到最适pH和pH稳定性趋近于生理中性pH 7.4的突变体Gly264Pro，其第264位甘氨酸Gly突变为脯氨酸Pro。
所述的精氨酸脱亚胺酶突变体的构建方法，包括如下步骤：
以来自于大肠杆菌宿主携带的精氨酸脱亚胺酶基因的重组质粒为模版，以带有突变位点的寡聚核苷酸序列为引物，进行反向PCR，扩增突变质粒全长；使用Dpn Ⅰ限制性内切酶进行消化；取经过Dpn Ⅰ限制性内切酶处理的PCR产物热机转化E.coli DH5α，涂布于含有卡那霉素抗性的固体LB培养基上培养；挑取固体LB培养基上的单菌落，接入含卡那霉素抗性的液体LB培养基中，提取质粒，测序；将测序结果正确的质粒转化入大肠杆菌E.coli BL21(DE3)感受态细胞中，获得突变体基因工程精氨酸脱亚胺酶。
所述精氨酸脱亚胺酶突变体的应用，所述的精氨酸脱亚胺酶突变体用于药用抗肿瘤活性及相关药理活性研究。
所述的氨基酸发生突变位于精氨酸脱亚胺酶蛋白结构的内部，所述的突变可以增加蛋白疏水相互作用。
所述的氨基酸发生突变的是精氨酸脱亚胺酶第264位的甘氨酸替换为脯氨酸，所得单突变体命名为Gly264Pro。
本发明提供一种所述精氨酸脱亚胺酶突变体的构建方法，具体步骤如下：
1、构建一株以pET-28a-c(+)为表达载体、在表达宿主大肠杆菌BL21(DE3)中表达的精氨酸脱亚胺酶基因工程重组菌；
2、设计突变引物，通过反向PCR对精氨酸脱亚胺酶基因进行定点突变，获得含突变精氨酸脱亚胺酶基因序列的重组载体；
3、将突变后的重组载体通过热激转化进入大肠杆菌BL21(DE3)中，并诱导表达，收集菌体，超声破碎细胞后使用Ni-NTA进行蛋白分离纯化，获得突变精氨酸脱亚胺酶。
本发明提供的精氨酸脱亚胺酶突变体最适pH和pH稳定性均向生理中性方向偏移，其最适pH从原始的5～5.5，扩大为5.5～7.5，pH稳定范围从5.5～6.5，偏移到6.5～7.5。解决了野生型的精氨酸脱亚胺酶在生理pH7.4条件下催化活性和稳定性低的问题，为该酶在医药领域的应用创造好的条件。
本发明所用的精氨酸脱亚胺酶的基因arcA来自于一株可生产瓜氨酸的粪肠球菌，该菌株编号CCTCC NO:M 2011465，保藏于中国典型培养物保藏中心，地址：中国武汉、武汉大学，命名为SK32.001(Enterococcus faecalis SK32.001)，在中国专利CN 102433290 A中已经公布。
附图说明
图1：重组质粒pET-28a-ADI的构建图谱。
图2：野生酶和突变酶的最适pH变化图。
图3：野生酶和突变酶的pH稳定性变化图。
具体实施方式
以下通过实施例来进一步阐明本发明，下列实施例用于说明目的而非用于限制本发明范围。
材料与试剂：所用的限制性内切酶、Solution Ⅰ连接酶、PCR试剂等均购于TaKaRa宝生物公司；质粒提取试剂盒、基因组提取试剂盒、琼脂糖纯化试剂盒、大肠杆菌DH5α、BL21(DE3)菌株、引物均购于生工生物工程(上海)有限公司；其他试剂均为国内或国外购买的分析纯试剂。
实施例1：重组质粒的构建
将Enterococcus faecalis SK32.001培养至指数生长中期，取2mL菌液10000r/min离心10min弃上清，溶菌酶处理30min后按照试剂盒说明提取基因组DNA。
设计如下的引物来用于arcA的扩增：
FADI-2：5’-CGCGGATCCATGAGTCATCCAATTAATGT-3’(含BamH I酶切位点)，
RADI-2：5’-CCGCTCGAGTTAAAGATCTTCACGGT-3’(含Xho Ⅰ酶切位点)，
PCR扩增条件：95℃变性3min，30个循环(95℃ 30s，55℃ 30s，72℃ 210s)，最后72℃延伸2min。
扩增产物纯化后，用BamH I和Xho Ⅰ对PCR产物和载体pET-28a-c(+)进行双酶切，分别回收酶切产物，用Solution Ⅰ连接酶16℃下连接2h，热激转化到DH5α细胞中，待平板长出转化子后，挑取单菌落到LB培养基中，提取质粒，通过酶切验证重组质粒pET-28a-ADI。将质粒转化到BL21(DE3)细胞中，得到BL21(DE3)/pET-28a-ADI工程菌。
实施例2：定点突变
根据Enterococcus faecalis SK23.001中编码arcA的编码基因，进行引物设计。
G264P-正向引物：5’-CTTGGCTTTTGATATCCCTGAACATCGTAAATTC-3’，
G264P-反向引物：5’-GATATCAAAAGCCAAGATATTTTTGAATCCTA-3’，
其中下划线部分代表突变体基因编码的264位甘氨酸所对应的密码子。PCR扩增体系为：
10X Reaction Buffer 5
dNTP mix 1
正向引物(100ng/μL) 1.25
反向引物(100ng/μL) 1.25
模版pET-28a-ADI(10ng) 2
PfuTurbo DNA polymerase(2.5U/μL) 1
ddH2O 38.5
总体积 50
PCR扩增后，向反应液中加入1μL Dpn Ⅰ限制性内切酶(10U/μL)，在37℃下保温1h消除模板。将PCR产物转化至大肠杆菌DH5α细胞中，涂布平板，挑取单菌落到LB培养基，提取质粒，经测序得到正确突变质粒，将构建成功的突变质粒转化入大肠杆菌BL21(DE3)中，得到突变株BL21(DE3)/pET-28a-ADIG264P。
实施例3：野生酶及突变酶的表达纯化
挑取BL21(DE3)/pET-28a-ADI及pET-28a-ADIG264P单菌落分别于含0.5mmol/L卡那霉素的LB培养基中，37℃、200r/min培养12h后，转接至含0.5mmol/L卡那霉素的LB培养基中，在37℃、200r/min培养至OD600在0.5～0.7范围内，加入1mmol/L的IPTG在28℃、200r/min条件下诱导9h。
发酵液在10000r/min、4℃下离心10min后，弃上清，用磷酸盐缓冲液洗涤两次，加入15～20mL磷酸盐缓冲液使菌体悬浮，超声破碎15min(功率22W，破碎1 s，间歇2s)。在4℃、10000r/min条件下离心10min，收集上清液，即为粗酶液，用孔径0.22μm水系膜过滤。
用Binding Buffer对Ni2+螯合琼脂糖树脂柱进行预平衡；加入粗酶液，分别用Binding Buffer和Washing Buffer平衡；用Elution Buffer将酶洗脱下来，回收；将回收的酶液在透析缓冲液中透析后于4℃冰箱保存。
所涉及缓冲液配制：
1磷酸盐缓冲液(PB)：50mmol/L，pH5.5；
2BindingBuffer：50mmol/LPB，500mmol/LNaCl，pH7.0；
3Washing Buffer：50mmol/LPB，500mmol/LNaCl，pH7.0，50mmol/L咪唑；
4Elution Buffer：50mmol/LPB，500mmol/LNaCl，pH7.0，500mmol/L咪唑；
5透析缓冲液：50mmol/LPB，pH5.5，10mmol/LEDTA。
实施例4：野生酶及突变酶的最适pH和pH稳定性测定
酶活测定方法：0.5mL底物L-精氨酸(浓度为10g/L)，加入50mmol/L的PB缓冲液0.49mL于45℃保温5min，然后加入0.01mL的酶液在45℃下反应10min，煮沸10min终止反应，将反应液离心去上清测定瓜氨酸的含量。
酶活定义：1min内产生1μmol L-1瓜氨酸所需的酶量定义为1个酶活单位(U)。
瓜氨酸含量测定：高效液相色谱法：Angilent 1200；色谱柱：Hypersil ODS(5μm，4.0mm×250mm)；流动相A：水2L，三水合乙酸钠13g，三乙胺0.4mL，四氢呋喃5mL，pH7.2±0.5；流动相B：2L，三水合乙酸钠15g，水/甲醇/乙腈(体积比1:2:2)，pH7.2±0.5；流动相A与B梯度洗脱，总流速：1.0mL/min；柱温：40℃；进样量：10μL；检测器：紫外检测器；检测波长：338nm，发射波长360nm。
最适pH：将预保存在50mM、pH4.0～7.5的PB缓冲液的野生酶或突变酶置于45℃水浴中，反应10min后立即煮沸终止反应。pH稳定性：将相同浓度的野生酶及突变酶分别在pH为4.0～7.5的缓冲液中于4℃预保存12h，然后在45℃条件下测定酶活力。
得到的结果如附图2：突变酶与野生酶相比，有效pH作用范围拓宽并向中性偏移。如附图3：突变酶与野生酶相比，在偏中性的pH下保存更稳定。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
Figure PCTCN2017113162-appb-000001
Figure PCTCN2017113162-appb-000002
Figure PCTCN2017113162-appb-000003
Figure PCTCN2017113162-appb-000004
Figure PCTCN2017113162-appb-000005
Figure PCTCN2017113162-appb-000006
Figure PCTCN2017113162-appb-000007
Figure PCTCN2017113162-appb-000008
Figure PCTCN2017113162-appb-000009
Figure PCTCN2017113162-appb-000010
Figure PCTCN2017113162-appb-000011
Figure PCTCN2017113162-appb-000012
Figure PCTCN2017113162-appb-000013

Claims (9)

  1. 一种精氨酸脱亚胺酶突变体，其氨基酸序列如SEQ ID No.1所示。
  2. 编码权利要求1所述的精氨酸脱亚胺酶突变体的基因，其核苷酸序列如SEQ ID No.2所示。
  3. 一种重组质粒，其特征在于，其包含了权利要求2所述的基因。
  4. 一种宿主细胞，其特征在于，其含有如权利要求2所述的基因，或含有如权利要求3所述的重组质粒。
  5. 权利要求1所述的精氨酸脱亚胺酶突变体的构建方法，其特征在于，包括如下步骤：
    以来自于大肠杆菌宿主携带的精氨酸脱亚胺酶基因的重组质粒为模版，以带有突变位点的寡聚核苷酸序列为引物，进行反向PCR，扩增突变质粒全长；使用Dpn Ⅰ限制性内切酶进行消化；取经过Dpn Ⅰ限制性内切酶处理的PCR产物热激转化E.coli DH5α，涂布于含有卡那霉素抗性的固体LB培养基上培养；挑取固体LB培养基上的单菌落，接入含卡那霉素抗性的液体LB培养基中，提取质粒，测序；将测序结果正确的质粒转化入大肠杆菌E.coli BL21(DE3)感受态细胞中，获得突变体基因工程精氨酸脱亚胺酶。
  6. 权利要求1所述精氨酸脱亚胺酶突变体的应用，其特征在于，所述的精氨酸脱亚胺酶突变体用于药用抗肿瘤活性及相关药理活性研究。
  7. 权利要求1所述精氨酸脱亚胺酶突变体在制备用于抑制精氨酸缺陷性肿瘤、乳腺癌、肝癌细胞的药物中的应用。
  8. 权利要求1所述精氨酸脱亚胺酶突变体在制备用于治疗白血病的药物中的应用。
  9. 权利要求1所述精氨酸脱亚胺酶突变体在于用于瓜氨酸的生产应用。
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