CN114752617A

CN114752617A - Method for constructing recombinant expression vector, engineering bacterium and expression system

Info

Publication number: CN114752617A
Application number: CN202210638496.XA
Authority: CN
Inventors: 毛德才; 罗晓林; 徐亚欧; 张玉; 陈莉敏; 安添午; 赵洪文
Original assignee: Southwest Minzu University; Sichuan Academy of Grassland Science
Current assignee: Southwest Minzu University; Sichuan Academy of Grassland Science
Priority date: 2022-06-08
Filing date: 2022-06-08
Publication date: 2022-07-15

Abstract

The invention relates to gene engineering, and provides a method for constructing a recombinant expression vector containing a yak manganese superoxide dismutase gene in order to improve the yield of superoxide dismutase, which comprises the following steps: (1) designing an amplification primer of the Mn-SOD according to the Mn-SOD gene sequence, and amplifying the Mn-SOD; (2) purifying the amplification product obtained in the step (1), connecting the purified amplification product to a transfer vector, transferring the purified amplification product to an Escherichia coli host cell DH5 alpha, and sequencing plasmids in Escherichia coli; (3) carrying out enzyme digestion on the plasmid obtained in the step (2) and the ligation vector by using restriction enzyme so as to connect the Mn-SOD fragment into the ligation vector; (4) transforming the product of the step (3) into an escherichia coli competent cell DH5 alpha, and sequencing after successful transfection; obtaining the expression vector. The invention also provides a genetic engineering bacterium matched with the expression vector and a high-efficiency expression system of the recombinant yak Mn-SOD protein. By the method, the manganese superoxide dismutase can be efficiently expressed, and conditions are provided for further large-scale production and utilization of yak Mn-SOD.

Description

Method for constructing recombinant expression vector, engineering bacterium and expression system

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a method for constructing a recombinant expression vector containing a yak manganese superoxide dismutase gene, engineering bacteria and a high-efficiency expression system of recombinant yak Mn-SOD protein.

Background

Yaks (Bos muscles or Bos grunniens or yak) are mainly distributed in Qinghai-Tibet plateau and adjacent high mountain areas, are precious resources of important life sources such as meat, milk, skin and the like, enjoy the reputation of a 'boat of plateau' and have good adaptability to extreme environments such as high altitude, low oxygen in air, strong ultraviolet radiation and the like. Research shows that the antioxidant system plays an important role in the moderate adaptation of animals and plants to the environment.

Superoxide dismutase (SOD) is an oxidoreductase that is widely present in various organisms in the natural world and specifically removes Superoxide anion (O) radicals generated by biological oxidation in the organisms^2-) And the like, thereby effectively defending the active oxygen from poisoning organisms, resisting aging and inhibitingTumor, inflammation and radiation resisting, and ischemia re-injection injury relieving effects. The manganese superoxide dismutase is mainly present in mitochondria, is the first defense line for preventing the mitochondria from being damaged by superoxide anions, can rapidly remove the superoxide anion free radicals generated during the electron transfer of the mitochondrial respiratory chain, protects the mitochondria from being damaged by the superoxide anions, and maintains the integrity and normal functions of subcellular organelles such as the mitochondria and the like.

Sufficient Mn-SOD sources are the precondition of wide application, but the natural Mn-SOD sources are limited, the purification process is complex, the environmental pollution pressure is large, the sample recovery rate is low, the acquisition cost is high, and the requirements are difficult to meet. Therefore, the utilization of genetic engineering means to obtain high-expression recombinant Mn-SOD protein has been the focus of research, but the realization of high-efficiency expression system of recombinant protein is a technical difficulty to be urgently broken through in the field.

Content of application

The first purpose of the invention is to provide a method for constructing a recombinant expression vector containing a yak manganese superoxide dismutase (Mn-SOD) gene and engineering bacteria thereof.

The second purpose of the invention is to provide a high-efficiency expression system of recombinant yak Mn-SOD protein based on the method and the engineering bacteria, which can efficiently express the Mn-SOD protein.

The embodiment of the invention is realized by the following technical scheme: the high-efficiency expression system of the recombinant yak Mn-SOD protein is prepared by the following steps:

(1) designing an amplification primer of Mn-SOD according to a coding region sequence of a yak Mn-SOD gene (sequence number XM-014479669), wherein in an upstream primer Mn-SOD-F and a downstream primer Mn-SOD-R of a yak, the upstream primer Mn-SOD-F contains a restriction enzyme NdeI restriction site, and the downstream primer Mn-SOD-R contains a restriction enzyme HindIII restriction site and a His label, and chemically synthesizing to obtain a primer sequence.

(2) Extracting yak liver RNA, performing reverse transcription to synthesize a first cDNA chain, and performing reverse transcription polymerase chain reaction (RT-PCR) to amplify yak Mn-SOD by taking the cDNA as a target and Mn-SOD-F and Mn-SOD-R as primers. Wherein, the RNA extraction adopts a conventional Trizol method, and the total RNA of the yak liver is extracted under the condition of RNAase treatment in the extraction process.

(3) And (3) purifying the RT-PCR product, connecting the RT-PCR product to a pMD18-T vector, transforming the product to an escherichia coli host cell DH5 alpha, and confirming the plasmid with correct sequencing through bacterial liquid PCR, plasmid restriction enzyme identification and plasmid sequencing, wherein the plasmid with correct sequencing is named as Y-Mn-SOD-T.

(4) The plasmids Y-Mn-SOD-T and pET22b (+) were digested with restriction enzymes NdeI/HindIII, and the objective fragment Mn-SOD was ligated to pET22b (+). Wherein, except for the T4 ligase for the ligation and the T4 ligation buffer solution, other methods for the digestion and ligation are the same as the corresponding methods in the step (3).

(5) And transforming the ligation product into host bacteria escherichia coli competent cells DH5 alpha, carrying out PCR identification and plasmid enzyme digestion identification after overnight culture at 37 ℃, and obtaining the yak Mn-SOD-pET22b (+) expression vector after correct sequencing. Wherein the conversion method, the reaction system and the reaction conditions are the same as above.

(6) The yak Mn-SOD-pET22b (+) plasmid is transmitted to escherichia coli BL21(DE3) host bacteria to form Mn-SOD-PET22b (+) -BL21 genetic engineering bacteria. Wherein the infection system and conditions are the same as those in the above step (3) except that the recombinant gene host strain is BL21(DE 3).

(7) Adding a proper amount of inducer Isopropyl-beta-D-thiogalactopyranoside (IPTG) into the cultured engineering bacteria to induce the expression of the recombinant yak Mn-SOD, and then identifying the expression product of the recombinant yak Mn-SOD. The identification method of the recombinant yak Mn-SOD expression product comprises an SDS-PAGE electrophoresis method and an immunoblotting hybridization Western Blot method.

(8) The invention also provides fermentation, crude protein extraction and enzyme activity determination of the recombinant yak Mn-SOD engineering bacteria.

The technical scheme of the embodiment of the invention at least has the following advantages and beneficial effects: through the efficient expression system of the recombinant yak Mn-SOD protein, high expression of the yak Mn-SOD is realized in escherichia coli, and the yak Mn-SOD has activity after simple renaturation, so that the premise is provided for further optimizing renaturation conditions, developing a purification process and carrying out large-scale production and utilization.

Drawings

FIG. 1 shows the result of agarose gel electrophoresis detection of the yak Mn-SOD RT-PCR amplification product of example 1 of the present invention;

FIG. 2 shows the difference between the Mn-SOD amino acid sequence of yak in example 2 and other species;

FIG. 3 is the SDS-PAGE detection result of prokaryotic expression of yak Mn-SOD in example 3 of the present invention;

FIG. 4 is a Western Blot result of prokaryotic expression of yak Mn-SOD in example 3 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

Example 1

Constructing a yak Mn-SOD recombinant vector.

1. Design of primers

Referring to yak Mn-SOD cDNA sequence number XM _014479669 in GenBank database, primers are designed by Primer5.0 software, NdeI restriction enzyme site and HindIII restriction enzyme site are added at two ends respectively, and the tail end is provided with a His tag, wherein an upstream primer: 5'-GCGCATATGATGCAGCTGCACCACAGCAAGC-3' (NdeI), downstream primer: 5'-CCGGATCCTTAATGGTGATGGTGATGATGCTTGCTGCAAGCCGTGTATCG-3' (HindIII). After design, the primers are synthesized by the company of Biotechnology engineering (Shanghai) GmbH (short for Biotechnology).

2. Yak total RNA extraction

Extracting yak liver total RNA by using TRizol (ThermoFisher SCIENTIFIC TRIZOL) according to conventional method, and detecting concentration and purity with spectrophotometer, wherein OD is₂₆₀/OD₂₈₀At 1.85-2.05, the integrity of the extracted RNA is good, and the next experiment is carried out.

RT-PCR amplification

Taking yak liver total RNA as a template and Oligo-T as a primer, and utilizing a YEASEN HifairIII first strandThe residual genomic DNA was removed in a 200. mu.L RNA free PCR tube using 20. mu.L of the reaction solution prepared as a 5 Xg DNA digestion mixture of 3. mu.L, 2. mu.g total yak RNA, and RNA free ddH₂Adding O to 15 mu L, lightly blowing and uniformly mixing by using a pipette, adding 5 mu L of 4 xHifaiIII Supermix plus at 42 ℃ for 2 minutes, and carrying out reverse transcription reaction after lightly mixing, wherein the reverse transcription reaction conditions are 5min at 25 ℃, 15min at 55 ℃ and 5min at 85 ℃. After the first cDNA strand was synthesized, 25.0. mu.L of the total PCR amplification reaction system was prepared using cDNA as a template as follows: 2 XM 5 HiPer plus Taq HiFi PCR mix: 12.5 mu L of yak cDNA, wherein the upstream and downstream primers Mn-SOD-F and Mn-SOD-R are respectively 0.5 mu L, 1.0 mu L of yak cDNA is formed, and deionized water ddH is added₂O10.5 mu L; the PCR reaction procedure was as follows: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 45s, annealing at 61 ℃ for 30s, extension at 72 ℃ for 1min, and total design for 35 cycles; further extension at 72 deg.C for 5 min; storing at 4 ℃. The PCR product was detected by 1.0% agarose gel electrophoresis, and the results are shown in FIG. 1 (in FIG. 1, lane M is DNA standard molecular weight DL2000, the left side number indicates the nucleotide size corresponding to each band; lane 1 is yak Mn-SOD RT-PCR amplification result, the right side number indicates the expected size), and a 531bp band was obtained, which was identical to the expected size.

4. Cloning of Yak Mn-SOD

(1) Cutting Mn-SOD PCR product fragments from agarose gel by using a biological DNA gel recovery kit, weighing, adding a Buffer Solution B2 with the weight being 3 times of that of the gel block, dissolving for 10 minutes at 55 ℃ and 450rpm, transferring the sol Solution into an adsorption column, centrifuging for 1 minute at 10000rpm, discarding the liquid in a collection tube, adding Wash Solution 500 mu L, centrifuging for 1 minute at 12000rpm (repeating for 1 time), centrifuging for 2 minutes at 12000rpm by using an air adsorption column, adding 30 mu L of precipitation Buffer in the center of the adsorption column, standing for 3 minutes at room temperature, centrifuging for 1 minute, and storing the DNA Solution.

(2) Connecting the recovered PCR product with TaKara PMD18T carrier, wherein the method is to construct a total connection reaction system of 10 μ L: the ligation reaction system comprises 1 mu L of pMD18-T, 3 mu L of Mn-SOD PCR product and ddH₂mu.L of O1 and 5 mu.L of connecting liquid, and after overnight at 16 ℃, taking the connecting product for transformation, wherein the transformation method comprises the following steps: taking 5 mu L of the ligation product, adding melted competent cell YEASEN DH5 alpha 100 mu L,ice-bath for 30 minutes; heat shock at 42 deg.c for 90 sec; rapidly ice-bathing for 3 minutes, and adding 150 mu L of LB liquid culture medium; incubation at 37 ℃ for 30 min; the transformation products were spread evenly on LB solid medium containing antibiotics using a glass rod and cultured overnight at 37 ℃. Wherein the LB liquid culture medium is prepared from 10g of tryptone, 5g of yeast extract and 10g of NaCl, deionized water is added to adjust the pH value to 7.0 and the volume to 1000mL, and the mixture is sterilized for 30 minutes under high pressure at 121 ℃; the LB solid culture medium is prepared by adding 15g of agarose into each 1000mL of LB liquid culture medium, mixing uniformly, and sterilizing for 30 minutes at 121 ℃; adding ampicillin to a final dose of 0.1mg/mL when the temperature is reduced to a proper temperature, mixing uniformly, pouring into a sterile plate, cooling, storing at 4 ℃, and adding a small amount of sterile IPTG and X-Gal before use.

In order to identify the cloning effect, white spots are picked to LB liquid culture medium, after 16 hours at 37 ℃ and 200rpm, bacteria liquid is picked for PCR identification, plasmid DNA is extracted from the correctly identified bacteria liquid, and the bacteria liquid is sent to biological sequencing. The sequencing result is SEQ ID 1, the corresponding amino acid sequence is SEQ ID2, the sequencing result is compared with NCBI BLAST, and the sequence is designed by combining a primer, and the result is consistent with the expected sequence, thereby indicating that the cloning of the yak Mn-SOD is successful.

Example 2

This example is to construct yak Mn-SOD gene engineering bacteria.

1. Joining the target fragment to an expression vector

The expression vector used in the invention is pET22b (+), the restriction enzymes and the enzyme cutting positions designed and used are NdeI and HindIII, the yak Mn-SOD plasmid and pET22b (+) are respectively double-cut by NdeI and HindIII (NEB), the reaction system is 50 muL, wherein 10 xBuffer 5 muL, the plasmids 10 mu L, NdeI/HindIII respectively 1 muL, and ddH is added₂O to 50. mu.L under the reaction conditions of 37 ℃ overnight, and the desired fragment was recovered by the above-mentioned conventional method. Adding a proper amount of enzyme digestion product yak Mn-SOD, an expression vector, T4 ligase, T4 connecting buffer solution and ddH₂O was metered to 10. mu.L, mixed well and ligated overnight at 16 ℃ and the ligation product was transformed into DH 5. alpha. competent cells by the method described above and cultured overnight in a37 ℃ incubator. Selecting white colony, inoculating into 15mL sterile centrifuge tube with 4mL LB culture medium, culturing at 37 deg.C overnight with 200rpm shaking table, performing PCR identification with culture solution, and performing PCR identification methodAs above. Detection was then performed on a 1% agarose gel. The detection result is consistent with the expected size.

2. Identification and analysis of fragments of interest

In order to further determine whether the establishment of the yak Mn-SOD recombinant is successful, the plasmid is extracted from the correct bacterial liquid identified by PCR and sent to biological sequencing. The sequencing results are completely consistent with the expected sequence through comparative analysis, which indicates that the yak Mn-SOD/pET22b (+) recombinant strain is successfully constructed in the invention, and in order to further analyze the specificity of the yak Mn-SOD gene and the conservative relationship among other species, human, common cow, goat, sheep, panda and chicken Mn-SOD sequences are selected to be compared with the yak Mn-SOD gene sequence, and the results are shown in figure 2 (in figure 2, Homo sapiens or Homo sapiens indicates human, Ailuropoda melaleueuca or Ailuropoda _ melaleueuca indicates panda, Bos grunniens or Bos _ ungren indicates yak, Bos taurus or Bos _ taurus indicates common cow, Capra hircus or Capra _ hircus indicates common cow, Ovis aries or Ovis _ aries indicates sheep, Gallus gagruus or Chicken _ gallinarus indicates number of nucleotide, and the number of common cow Mn-SOD sequences can be respectively represented by the figure 2, the number of common cow, goat and goat, and the number of nucleotide can be respectively represented by the figure 2, the sequence of the yak Mn-SOD 99.81%, 98.31%, 98.49%, 90.02% and 80.79% of the corresponding encoded amino acid sequences, which differ by 12, 0, 1, 12, 18, respectively, in particular, the yak Mn-SOD amino acid sequence involved in the present invention differs by human (A19T, R24Q, E28A, Q68K, A82D, V92A, Q110A, R112, S119A, A A, T173A, S175A), common cow (0), goat (Q68A), sheep (Q68A), giant panda (A19, R24A, E28A, Q68, A83 4, V92, Q110, R112, S A, S119A, T175, A, S175, S A, S175, A, S175, A, S175, A, S55, S175, A, S55, S175, A, S13, A, S55, A, S175, S13, A, S175, A, S175, S13, A, S13, A, S175, S13, A, S13, A, S13, A, S175, A, S13, A, S13, A, S21, S13, A, S13, S21, S13, S21, S13, S21, S13, S21, S13, A, S13, K and other species with higher than the species, K and other species, K and S13, K and chicken species, K and S13, K3, K4, K7, K4, K13, K7, A, K and S13, K and chicken species, A, K, particularly, the nucleotide sequence consistency with common cattle is 99.81 percent, but the coded amino acid sequence consistency is 100.00 percent, which indicates that the difference of the Mn-SOD nucleotide sequences of the yaks and the common cattle related by the invention is nonsense variation.

3. Establishment of recombinant yak Mn-SOD engineering bacteria

After extracting Mn-SOD-PET22b (+) plasmid, transforming it into Escherichia coli BL21(DE3) by the above method, picking recombinant, culturing overnight in LB liquid culture medium containing ampicillin, taking part of culture liquid to extract plasmid by the above method, and sequencing to confirm the accuracy of recombinant.

Example 3

This example shows the induced expression and identification of recombinant yak Mn-SOD.

1. Recombinant yak Mn-SOD induced expression

Mixing the yak Mn-SOD-pET22b (+) engineering bacterium liquid with 4mL LB liquid culture medium in a ratio of 1: 50-1: culturing at 100 proportion, 37 deg.C, 200rpm, when OD₆₀₀When the concentration was 0.5 to 1.0, IPTG was added to 0.1mM, and the culture was continued for 5 hours, while BL21 E.coli and BL 21-transferred pET22b (+) vector were used as controls, and the expression cells were collected at 5000rpm and 4 ℃.

2. SDS-PAGE identification of recombinant yak Mn-SOD induced expression

The collected Mn-SOD expressing bacteria were detected by 12% SDS-PAGE electrophoresis. The detection result is shown in FIG. 3 (in FIG. 3, lane M is a protein non-prestained Marker III, and the left side of lane M indicates the corresponding molecular weight of each band; lane 1, lane 2, lane 3, lane 4, lane 5, lane 6 and lane 10 are IPTG-induced Mn-SOD-pET22B (+) -BL21 engineered bacteria expression proteins with different concentrations, wherein, the band in the red frame is recombinant yak Mn-SOD expression protein; lane 7 is BL21 bacteria expression protein (control); lane 8 is pET22B (+) -BL21 bacteria expression protein (control); lane 9 is other expression protein (control)), compared with BL21 E.coli sample, pET22B (+) vector sample transferred into B21 and uninduced engineered bacteria sample band, BL21 E.coli sample and pET22B (+) vector sample transferred into B21 do not express, but the expression of BL21 E.coli sample and pET 22-SOD-pET 22B (+) vector sample transferred into B21 respectively show about 21Kda and are obviously different in size, the size was consistent with expectations. Therefore, in SDS-PAGE electrophoresis detection, a band specifically expressed in the escherichia coli may be recombinant yak Cu/Zn-SOD protein, but needs to be further identified.

3. Western Blot identification of recombinant yak Mn-SOD induced expression

The invention relates to an identification method of yak Mn-SOD protein, which is characterized in that the size and the expression level of yak Mn-SOD protein are preliminarily judged by 12% SDS-PAGE electrophoresis, and particularly, an immunoblotting method (Western Blot which contains or is abbreviated as letters and is not in case) is applied for identification, namely His is used as a specific antibody to detect whether Mn-SOD with a label is specifically expressed or not, and the method comprises the following steps: the expression proteins of BL21, pET22b (+) -BL21 and yak Mn-SOD-pET22b (+) -BL21 with the same amount are subjected to 12% SDS-PAGE electrophoresis, then gel is cut, a PVDF membrane is utilized, and after conventional treatment, a sandwich is made for electrophoretic transfer. Subsequently, the cells were blocked with 5% skim milk, washed 5 times for 10 minutes each on a conventional 1 × PBST shaker, and after washing, the mouse anti-His antibody diluted 1:1000 was added, and after overnight at 4 ℃, washed 5 times for 10 minutes each with 1 × PBST wash solution; after washing, ECL chemiluminescence liquid with the same volume as AB is added, a chemiluminescence detection system of a GENEBOX gel imaging system is used for detection, the detection result is shown in figure 4 (in figure 4, 1 is the identification result (contrast) of the expression protein of BL21 bacteria, 2 lanes are the identification result (contrast) of the expression protein of pET22B (+) -BL21 bacteria, and 3 is the identification result of the expression protein of yak Mn-SOD-pET22B (+) -BL21 engineering bacteria), the yak Mn-SOD-pET22B (+) sample has immunoblot spots, and the control groups BL21 and pET22 (+) -22B (+) -B21 do not have immunoblot spots, which indicates that the recombinant yak Mn-SOD prepared by the invention is specially expressed.

Example 4

This example is the extraction and activity determination of recombinant yak Mn-SOD engineering bacteria total protein.

1. Extraction of recombinant yak Mn-SOD engineering bacterium total protein

Weighing the collected yak Mn-SOD-pET22b (+) bacteria, adding a proper amount of 25Mm Tris-100mM NaCl buffer solution, ultrasonically cleaning, adding a small amount of 5-8M guanidine hydrochloride to completely dissolve the guanidine hydrochloride, centrifuging at 8000rpm for 10 minutes, renaturing at 100mM PBS (pH 7.6) and 4 ℃ for 48 hours, centrifuging at 5000rmp4 ℃ for 20 minutes, and taking the supernatant to determine the activity of the recombinant yak Mn-SOD.

2. Activity determination of recombinant yak Mn-SOD

The recombinant yak Mn-SOD renaturation solution obtained in the above way is measured by a Bradford protein quantitative reagent of a biological organism, BSA is used as a standard substance, and the concentration of the yak Mn-SOD recombinant protein is measured by a spectrophotometry method and a linear range (0 mu g/mL-10.0 mu g/mL) measurement method through a 1mL cuvette. The activity is measured by nitryl tetrazolium chloride (NBT) method according to the specification of Superoxide Dismutase (SOD) kit of Suzhou Ke Ming Biotechnology limited, specifically, preheating a spectrophotometer for more than 30 minutes, adjusting the wavelength to 560nm, and adjusting the zero with distilled water; uniformly mixing the reagent II and the distilled water in equal volume according to the required amount; after the reagent is dissolved in 5mL of distilled water (used up within one week), the reagent is diluted with distilled water by 1:3 according to the required amount; before the determination, the first, third and fourth reagents are put in a water bath at 25 ℃ for more than 5 minutes; the following reagents were added sequentially to a 1.5mL EP tube:

TABLE 1 determination of SOD Activity of samples reagents were added sequentially

The SOD enzyme activity calculation method comprises the following steps:

percent inhibition was 100% (a control tube-a assay tube)/a control tube.

SOD activity (U/mg prot) [ inhibition percentage/(1-inhibition percentage) × V total volume of reaction ]/(V-like × Cpr) ═ 11.4 inhibition percentage/(1-inhibition percentage)/Cpr.

According to the determination, the activity of the Mn-SOD of the recombinant yak is 12U/mg. The yak Mn-SOD related by the invention realizes high expression in escherichia coli, has activity after simple renaturation, and provides a premise for further optimizing renaturation conditions, developing a purification process and carrying out large-scale production and utilization.

The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and various modifications and changes will occur to those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Sichuan province grassland scientific research institute

<120> method for constructing recombinant expression vector, engineering bacterium and expression system

<130> 2022.06.07

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 531

<212> DNA

<213> Artificial Synthesis

<400> 1

atgcagctgc accacagcaa gcaccacgcg gcctacgtga acaacctcaa cgtcgccgag 60

gagaagtacc gggaggcgct ggagaagggt gatgttacag ctcagatagc tctgcagcct 120

gcgttgaagt tcaacggtgg gggccatatc aatcacagca tcttctggac aaatctgagc 180

cctaacggtg gtggagaacc ccaaggggaa ttgctggaag ccatcaaacg tgactttggt 240

tcctttgcca aatttaagga aaagttgact gctgtatctg ttggtgtcca aggctccggt 300

tggggttggc tcggcttcaa taaggagcag ggacgcttac agattgctgc ttgttctaac 360

caggatcccc tgcaaggaac aacaggtctt atccccctgc tggggattga cgtgtgggag 420

catgcttatt accttcagta taaaaatgtc aggcccgatt atctgaaggc catttggaat 480

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<213> Artificial Synthesis

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Met Gln Leu His His Ser Lys His His Ala Ala Tyr Val Asn Asn Leu

1 5 10 15

Asn Val Ala Glu Glu Lys Tyr Arg Glu Ala Leu Glu Lys Gly Asp Val

20 25 30

Thr Ala Gln Ile Ala Leu Gln Pro Ala Leu Lys Phe Asn Gly Gly Gly

35 40 45

His Ile Asn His Ser Ile Phe Trp Thr Asn Leu Ser Pro Asn Gly Gly

50 55 60

Gly Glu Pro Gln Gly Glu Leu Leu Glu Ala Ile Lys Arg Asp Phe Gly

65 70 75 80

Ser Phe Ala Lys Phe Lys Glu Lys Leu Thr Ala Val Ser Val Gly Val

85 90 95

Gln Gly Ser Gly Trp Gly Trp Leu Gly Phe Asn Lys Glu Gln Gly Arg

100 105 110

Leu Gln Ile Ala Ala Cys Ser Asn Gln Asp Pro Leu Gln Gly Thr Thr

115 120 125

Gly Leu Ile Pro Leu Leu Gly Ile Asp Val Trp Glu His Ala Tyr Tyr

130 135 140

Leu Gln Tyr Lys Asn Val Arg Pro Asp Tyr Leu Lys Ala Ile Trp Asn

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Val Ile Asn Trp Glu Asn Val Thr Ala Arg Tyr Thr Ala Cys Ser Lys

165 170 175

Claims

1. A method for constructing a recombinant expression vector containing a yak manganese superoxide dismutase gene is characterized in that the yak manganese superoxide dismutase gene is named as Mn-SOD, and the method comprises the following steps:

(1) designing an amplification primer of the Mn-SOD according to the Mn-SOD gene sequence, and amplifying the Mn-SOD;

(2) purifying the amplification product obtained in the step (1), connecting the purified amplification product to a transfer vector, transferring the purified amplification product to an Escherichia coli host cell DH5 alpha, and sequencing plasmids in Escherichia coli;

(3) carrying out enzyme digestion on the plasmid obtained in the step (2) and the connecting carrier by using restriction enzyme so as to connect the Mn-SOD fragment into the connecting carrier;

(4) and (4) transforming the product of the step (3) into an escherichia coli competent cell DH5 alpha, and sequencing after successful transfection to obtain an expression vector.

2. The method for constructing the recombinant expression vector containing the yak manganese superoxide dismutase gene as claimed in claim 1, wherein the transfer vector in the step (2) is pMD18-T, and the plasmid successfully transfected in the step (2) is named as Y-Mn-SOD-T.

3. The method for constructing the recombinant expression vector containing the yak manganese superoxide dismutase gene as claimed in claim 1, wherein the ligation vector in the step (3) is pET22b (+), and the restriction enzymes are NdeI/HindIII;

in step (4), the plasmid successfully transfected was designated Mn-SOD-pET22b (+).

4. An engineering bacterium containing a recombinant expression vector of a yak manganese superoxide dismutase gene, which is characterized in that the expression vector obtained in any one of claims 1 to 3 is transfected into a host bacterium to form the engineering bacterium.

5. The engineered bacterium containing the recombinant expression vector of the yak manganese superoxide dismutase gene as claimed in claim 4, wherein the host bacterium is Escherichia coli BL 21.

6. A high-efficiency expression system of recombinant yak Mn-SOD protein is characterized in that an inducer is used for carrying out induction expression on the engineering bacteria obtained in the claim 5 to obtain an Mn-SOD expression product, and the expression product is identified.

7. The high-efficiency expression system of the recombinant yak Mn-SOD protein as claimed in claim 6, wherein the inducer is isopropyl-beta-D-thiogalactoside.

8. The high-efficiency expression system of the recombinant yak Mn-SOD protein according to claim 6, wherein the identification method comprises SDS-PAGE electrophoresis and/or Western Blot hybridization.