CN110117607A - A kind of recombinant vector and expression of ustilago zeae effect protein Pit2 gene - Google Patents

Abstract

The invention discloses a kind of recombinant vectors of ustilago zeae effect protein Pit2 gene, and the invention also discloses a kind of methods for expressing ustilago zeae effect protein Pit2 gene；The present invention constructs the prokaryotic expression carrier of ustilago zeae effect protein Pit2 gene, and a series of optimization is carried out for protein expression condition, finally obtain Pit2 soluble protein, as a result further research albumin crystal structure and biological characteristics lay the foundation, thinking is provided for the pesticide manufacturing of target for Pit2 for design, and then provides foundation for prevention and treatment ustilago zeae.

Description

A kind of recombinant vector and expression method of corn smut effect protein Pit2 gene

technical field

The invention relates to a recombinant vector and an expression method of a corn smut effector protein Pit2 gene, belonging to the field of biotechnology.

Background technique

Corn smut is one of the main diseases of corn, which can occur throughout the growth period of corn. However, corn smut can be cooked or eaten raw when it is young. It has a sweet taste, and it has a different flavor when fried. Regular consumption can prevent and treat liver system and gastrointestinal ulcers, and can help digestion and laxative. Corn smut contains 16 kinds of amino acids such as glutamic acid, lysine, alanine, arginine, methionine, threonine, and histidine. This corn smut fungus can be processed and used for medicine. Pick off the fresh spore pile or collect the mature honey pills. It is cold in nature and sweet in taste, which is beneficial to the liver and stomach. It can also cure neurasthenia and children. malnutrition.

During the confrontation between plants and pathogenic bacteria, the pathogenic bacteria will secrete some proteins into the host cells. These proteins, known as "effector proteins (Avr)", can suppress plant immune responses in various ways and promote pathogens to successfully infect plants. In recent years, there have been reports on the cloning of effector protein genes of pathogenic bacteria, such as blast fungus, linsein rust fungus, oomycete, tomato leaf mold, Fusarium oxysporum, rapeseed canker, barley moire and corn black powder The effector protein genes in bacteria have also been isolated and cloned successively.

Prokaryotic expression of genes is one of the common techniques for studying gene function. Prokaryotic expression of proteins often occurs in Escherichia coli due to expression restrictions caused by rare codons, and inclusion bodies are formed; when highly toxic proteins are expressed, protein expression will be inhibited, resulting in low expression yield; another problem that is easily overlooked is the Too high reducibility will lead to incorrect formation of disulfide bonds, resulting in insoluble expression products. In view of the above problems, the usual practice is to dissolve the inclusion bodies with urea, purify them under denaturation conditions, and then renature. However, this method has cumbersome steps and a low success rate. At present, the effector protein Pit2 gene of maize smut has been discovered, but how to express it in a soluble form is still a difficult problem in this field. The invention screens the conditions for the soluble expression of Pit2 protein by selecting different bacterial strains, different induction temperatures and the concentration of the inducer IPTG, laying a foundation for the study of gene function and protein crystals.

Contents of the invention

Aiming at the deficiencies of the prior art, the object of the present invention is to provide a recombinant vector and expression method of the effector protein Pit2 gene of smut in corn.

The technical scheme of the present invention is: a recombinant vector of the effector protein Pit2 gene of corn smut, the preparation method of the recombinant vector is as follows: extract corn RNA, and reverse transcribe the cDNA; amplify the Pit2 gene with the cDNA as a template; The amplified product of Pit2 was double-digested with BamHI and XhoI, and then inserted into the pET-28a expression vector that had been cut with the same double-digestion by DNA ligase to obtain the recombinant plasmid pET-28a-Pit2.

Using cDNA as a template, the following primers were used to amplify the Pit2 gene. The primer sequences are as follows:

Upstream primer: Pit2-F: GC GAATTC ATTCCGGTGCGTCGATCG underlined is the BamHI restriction site;

Downstream primer: Pit2-R: CG CTCGAG TTATTCCCAGATGACCACATCT underlined is the XhoI restriction site;

The upstream and downstream primers are shown in SEQ ID NO.3 and 4 respectively.

The present invention also provides a kind of method expressing corn smut effect protein Pit2 gene, comprises the following steps:

(1) Transform Escherichia coli cells with the recombinant vector described in claim 1 or 2 to obtain a recombinant prokaryotic expression strain expressing Pit2;

(2) Inoculate the recombinant prokaryotic expression strain into kanamycin liquid medium or kanamycin+chloramphenicol liquid medium, and cultivate the activated strain overnight;

(3) The activated recombinant prokaryotic expression strain was transferred to the kanamycin liquid medium, and IPTG was added after shaking culture to induce the expression of recombinant Pit2;

(4) After the induction is completed, the expressed Pit2 recombinant protein is recovered and purified.

Preferably, in step (3), when shaking culture to OD600=0.5, IPTG with a final concentration of 1.0 mM is added to induce culture at 25°C.

Preferably, the kanamycin in the kanamycin liquid medium is 50ug/mL.

Preferably, the kanamycin in the kanamycin+chloramphenicol liquid medium is 50ug/mL, and the chloramphenicol is 50ug/mL.

The beneficial effects of the present invention are: in order to obtain the Pit2 soluble protein, the present invention constructs the prokaryotic expression vector of the corn smut effector protein Pit2 gene, and performs a series of optimizations on protein expression conditions, and finds that when the induction temperature is 25°C, The expression level is the best; with the increase of IPTG concentration, the protein expression level increases gradually, and the optimal induction concentration is 1.0mM; at 25℃, the expression level is the best when the IPTG concentration is 1.0. After SDS-PAGE electrophoresis detection, the target band with a molecular weight of about 17kDa was obtained, which was the expected size of the Pit2 protein, and finally the Pit2 soluble protein was obtained. At the same time, the present invention uses a protein purification system to purify it, and the result lays the foundation for further research on the protein crystal structure and biological characteristics, provides ideas for the design of pesticide development targeting Pit2, and provides a basis for the prevention and treatment of corn smut.

Description of drawings

Fig. 1 is the map of pET-28a vector;

Figure 2 is a schematic diagram of the enzyme digestion results, 1 is the digested plasmid DNA, 2 is the BamHI and XhoI endonuclease digestion results, M is the DNA Marker;

Figure 3 is a protein peak diagram obtained by purification and desalting of Pit2 protein through the AKTA protein purification system;

Fig. 4 is the electrophoresis result figure after Pit2 protein purification;

Fig. 5 is the result figure of SDS-PAGE electrophoresis test optimal IPTG induction concentration;

Fig. 6 is the result figure of SDS-PAGE electrophoresis test optimal induction temperature;

Figure 7 is a western blot detection map.

Detailed ways

Below in conjunction with accompanying drawing and specific embodiment the invention is further introduced:

Example 1: Construction and Identification of Maize Ustilago Effector Protein Pit2 Gene Recombination Vector

1. Extract corn RNA and reverse transcribe cDNA

The corn smut material was taken, and the total RNA at the seedling stage was extracted with an RNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.), and cDNA was obtained by reverse transcription with a reverse transcription kit (Promega).

2. Using cDNA as a template to amplify the Pit2 gene;

Primers were designed to amplify the Pit2 gene using cDNA as a template. The nucleotide sequence of the Pit2 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the Pit2 gene is shown in SEQ ID NO.2.

The above primers are as follows:

Upstream primer: Pit2-F: GC GAATTC ATTCCGGTGCGTCGATCG (remove the signal peptide sequence) is underlined as the BamHI restriction site;

Downstream primer: Pit2-R: CG CTCGAG TTATTCCCAGATGACCACATCT underlined is the XhoI restriction site;

The upstream and downstream primers are shown in SEQ ID NO.3 and 4 respectively.

The PCR amplification system used for gene amplification of vector construction is as follows: 5× buffer 10 μL, FastPfuFly DNA Polymerase 1 μL (full gold), 2.5 mmol dNTPs 4 μL, cDNA template 0.5 μL, 10 μmol upstream and downstream primers 1 μL each, ddH ₂ O 32.5 μL.

The following are PCR amplification conditions: 95°C for 2min, 35 cycles (95°C for 20s, 58°C for 20s, 72°C for 30s-1min, 72°C for 5min.

3. Construction of Pit2 gene recombination vector

After the PCR product was purified, it was connected to the pET-28a vector. PCR products were detected using 1% agarose. The PCR products were recovered by referring to the instructions of the agarose gel recovery kit of Nanjing Novizan Biotechnology Co., Ltd. The recovered product was double-digested with BamHI and XhoI and then inserted into the pET-28a expression vector (expression vector map shown in Figure 1) by DNA ligase, and ligated at 16°C for 16 hours to construct pET-28a. 28a-Pit2 recombinant plasmid.

The recombinant plasmid was transformed into Escherichia coli strain DH5a, cultured on a 50ug/mL kanamycin solid plate at 37°C, and a single colony was selected and identified by enzyme digestion (the results of enzyme digestion are shown in Figure 2), and then passed through Beijing Qing The correctness of the sequence was verified by Sequencing at Kebio Technology Co., Ltd. Sequencing results showed that the sequence of the coding region of the Pit2 gene obtained was consistent with the prediction, indicating that the recombinant plasmid was successfully constructed and named pET-28a-Pit2, namely the recombinant vector.

Example 2: Induced expression of Pit2 protein

1. Obtain the recombinant prokaryotic expression strain of Pit2

The single clone successfully sequenced in Example 1 was selected and inoculated into 50ug/mL kanamycin liquid medium, cultured overnight at 37°C and 200rpm, and the pET-28a -Pit2 recombinant expression vector was extracted, and the recombinant expression vector was transformed into Escherichia coli expression strains BL21(DE3), JM109(DE3), BL21(DE3)pLysS, Tuner(DE3), Rosetta(DE3), and the expression of Pit2 protein was detected.

2. Cultivate the activated strain overnight

The above-mentioned recombinant prokaryotic expression strains were activated by culturing overnight. For example, transfer BL21(DE3), JM109(DE3), Tuner(DE3) strains to 50ug/mL kanamycin liquid medium, transfer Rosetta(DE3), BL21(DE3)pLysS strains to 50ug/mL In mL kanamycin+50ug/mL chloramphenicol liquid medium, cultivate the activated strain overnight at 37°C and 200rpm.

3. Induced expression

Transfer the activated recombinant prokaryotic expression strains to the liquid medium containing the corresponding antibiotics, for example, transfer the activated BL21(DE3), JM109(DE3), Tuner(DE3) strains to 50ug/mL kanamycin liquid In the culture medium, Rosetta (DE3), BL21 (DE3) pLysS strains were transferred to 50ug/mL kanamycin + 50ug/mL chloramphenicol liquid medium, cultured with shaking at 37°C until OD600 = 0.5, and then added the final IPTG with a concentration of 1.0 mM was induced and cultured at 25° C. for 10 hours to obtain the recombinant protein of Pit2.

4. Purification of Pit2 recombinant protein

After the induction is complete, recover and purify the expressed Pit2 recombinant protein, use the AKTA protein purification system for purification and desalting, preferably, use the AKTA purifier10 fast protein purification system for protein purification, the steps are as follows:

Since pET28a has 6 His coding sequences before the terminator, the protein obtained from the pET28a-Pit2 recombinant expression vector can be purified by affinity chromatography using Ni-NTA. Affinity chromatography is based on the specific affinity between proteins or biomacromolecules and ligands bound to the medium, because the matrix of the chromatography gel used for His-tags is connected with an NTA [(nitrilotriacetic acid) nitrogen triacetic acid ], can be combined with Ni ions, and Ni ions have an attractive force between the 6×His amino acids of the fusion protein, thereby distinguishing the fusion protein with the His-tags histidine tag from other proteins, the histidine residue The five-membered imidazole ring of the base is the key to the interaction between the protein and Ni ions. Therefore, when we elute with a high-concentration imidazole (Imidazole) solution (such as 400mM), the imidazole is convenient for the imidazole ring of the protein His-tags to compete for binding, and finally The fusion protein is eluted from the gel.

Specific operation: Take out the bacterial cells stored at -80°C, add Lysis buffer to resuspend, the resuspended bacterial solution is ultrasonically crushed in an ice bath, the power is 350W, the duty cycle is 50%, each cycle is 30s, and the total time 35min. The crushed homogenate was centrifuged at 1,2000 rpm for 30 min at 4°C. Adsorb the supernatant to the Ni-NTA adsorption column with a peristaltic pump, 5mL/min, repeat once, retain 20μL of the sample that passes through the adsorption column for protein gel electrophoresis; connect the Ni-NTA adsorption column to the rapid protein purification system, and perform Elution; collect the protein according to the UV absorption curve, and take a sample for protein gel electrophoresis detection.

Figure 3 is a peak diagram of protein purified by the AKTA protein purification system. The protein peak diagram of Pit2 protein purified by AKTA protein purification system.

Fig. 4 is an electrophoresis diagram of purified Pit2 protein. It is the result of expressing and purifying Pit2 protein at 25°C and IPTG concentration of 1.0 mM. 1 is the protein purified by Ni column affinity; 2 is the supernatant sample after the cell is sonicated; M is the protein marker; the arrow indicates the Pit2 protein. Figure 4 shows that under this condition, the Pit2 protein mostly appeared in the supernatant and was expressed in a soluble manner. The purity of the Pit2 protein purified by the AKTA protein purification system met the requirements and could be used in subsequent experiments.

Example 3: Verification of Pit2 protein-induced expression conditions

1. Obtain the recombinant expression strain of Pit2

The single clone successfully sequenced in Example 1 was selected and inoculated into 50ug/mL kanamycin liquid medium, cultured overnight at 37°C and 200rpm, and the pET-28a -Pit2 recombinant expression vector was extracted, and the recombinant expression vector was transformed into Escherichia coli BL21 (DE3) strain, spread on a plate containing 50ug/mL kanamycin and cultured overnight at 37°C, and a single colony was picked and inoculated to 50ug/mL In kanamycin liquid culture medium, cultivate overnight at 37°C and 200rpm, add sterilized 50% glycerol to the bacterial liquid, mix well and store in a -80°C refrigerator to obtain a prokaryotic expression strain expressing Pit2.

2. Determination of the optimal IPTG concentration for Pit2 gene induction

Inoculate the above-mentioned recombinant prokaryotic expression strain BL21(DE3) into 50ug/mL kanamycin liquid medium, and cultivate the activated strain overnight at 37°C and 200rpm. The activated recombinant prokaryotic expression strain was then transferred to 50ug/mL kanamycin liquid medium, and cultured with shaking at 37°C until OD600=0.5, and then added IPTG with final concentrations of 0.4, 0.8, 1.0, and 1.2mM respectively. Induce culture at 25°C for 4-16 hours (take uninduced bacteria as negative control). After induction, centrifuge at 1,2000rpm for 2min at 4°C to collect the bacteria, suspend the bacterial pellet and the negative control in 2mL pre-cooled PBS buffer (add 1mM PMSF before use), and disrupt the cells with an ultrasonic breaker, 10sec each time, a total of 10 Centrifuge at 1,2000rpm for 10min, take the supernatant, detect by SDS-PAGE electrophoresis, test the optimal IPTG induction concentration, the results are shown in Figure 5, the optimal IPTG for Pit2 gene expression is 1.0mM.

3. Determination of the optimum temperature for induction and expression of Pit2 gene

Inoculate the above-mentioned recombinant prokaryotic expression strain BL21(DE3) into 50ug/mL kanamycin liquid medium, and cultivate the activated strain overnight at 37°C and 200rpm. The activated recombinant prokaryotic expression strain was transferred to 50ug/mL kanamycin liquid medium, and cultured with shaking at 37°C until OD600=0.5, adding IPTG with a final concentration of 1.0mM, respectively at 16, 20, 25, and 30 , and 37 ° C induction culture for 8-16 hours (take pET-28a bacteria as negative control). After induction, centrifuge at 1,2000rpm for 2min at 4°C to collect the bacteria, suspend the bacterial pellet and the negative control in 2mL pre-cooled PBS buffer (add 1mM PMSF before use), and disrupt the cells with an ultrasonic breaker, 10sec each time, a total of 10 Centrifuge at 1,2000rpm for 10min, take the supernatant, detect by SDS-PAGE electrophoresis, and test the optimal induction temperature. The results are shown in Figure 6. The optimal induction temperature for Pit2 gene expression is 25°C.

Example 4: Western blot detection of Pit2 recombinant protein

(1) Extract the purified Pit2 protein:

1. Cultivate the E. coli expression strain containing the pET-28a-Pit2 plasmid, and induce expression with IPTG;

2. Add protein lysate (50mM Tris pH 8.0, 120mM NaCl, 1mM DTT, 0.5% NP-40, add 1mM PMSF, 1mM DTT before use), break the cells with ultrasonic waves, collect supernatant by centrifugation, and place on ice for 20min;

3. Centrifuge at 15,000g at 4°C for 5min, discard the precipitate;

4. Take 200 μL supernatant and add 5×Loading Buffer, shake and mix well, and freeze the remaining samples at -80°C;

5. Boil in water for 5 minutes, ice bath, centrifuge at 12,000 rpm for 5 minutes, absorb 15 μL of the supernatant for protein electrophoresis, and freeze the remaining samples at -20°C;

6. To prepare SDS-PAGE gel, first use 90V for electrophoresis for 20 minutes, and when the sample enters the separation gel, use 150V for electrophoresis, and stop electrophoresis when bromophenol blue runs out of the separation gel.

(2) transfer film

Use a paper knife to cut out a PVDF membrane slightly larger than the glue (for the PVDF membrane, first soak it in methanol for 30 seconds, then soak it in ddH ₂ O for 3-5 minutes, and then put it in the transfer buffer). into the transfer electrophoresis tank. Pour into the transmembrane buffer, 4°C, 100V 90min.

(3) closed

After the membrane transfer was completed, the PVDF membrane was taken out, placed in a PBST solution with 5% skimmed milk powder, and blocked on a shaker for 2 hours.

(4) Primary antibody incubation

Discard the blocking solution, add Anti-His antibody (1:5000), and incubate on a shaker for 12 hours.

(5) Secondary antibody incubation

Pour off the primary antibody, wash the PVDF membrane 3 times with 1×PBST, 5 min each time; add the corresponding secondary antibody (goat anti-mouse), and incubate for 1 h on a shaking table.

(6) ECL Exposure

Discard the secondary antibody, wash the PVDF membrane 3 times with 1×PBST, 5min each time; add HRP chemiluminescence substrate solution on the PVDF membrane, and detect protein expression signal on Tianneng 5200 chemiluminescence detector (as shown in Figure 7) , it can be seen from the results that the Pit2 protein was successfully expressed.

List of nucleotide series:

SEQUENCE LISTING

sequence listing

<110> Guizhou University

<120> A recombinant vector and expression method of the effector protein Pit2 gene of corn smut

<160> 4

<210>1

<211>363

<212>DNA

<213> Corn smut effector protein Pit2

<400>1

ATGCTGTTTC GCTCAGCCTT TGTTCTGCTC ATCGTGGCCT TTGCAAGTGC ATGCCTGGTG 60

CAACATGTTC AAGCTAAGCA GATTCCGGTG CGTCGATCGC TCTCTACCGA TGCCTCAATG 120

AGCTCGGCTG CTGGCAAGCT CAACCGGAGA TGGTGGTTCG GCTTCACAGG TTCGCTCGGC 180

AAGGAACCTG ACAACGGCCA AGTACAGATC AAGATCATCC CAGACGCGCT CATCATCAAG 240

AATCCGCCTG CCAACAAAGA CGATCTGAAC AAGCTAATCG AAAACCTAAA ACGCAAGCAC 300

CCAAGATTCA AGACGGTGGT CATGCCGACA GATCCTAACG GAGATGTGGT CATCTGGGAA 360

TAA 363

<210>2

<211>120

<212>PRT

<213> Corn smut effector protein Pit2

<400>2

MLFRSAFVLL IVAFASACLV QHVQAKQIPV RRSLSTDASM SSAAGKLNRR WWFGFTGSLG 60

KEPDNGQVQI KIIPDALIIK NPPANKDDLN KLIENLKRKH PRFKTVVMPT DPNGDVVIWE 120

<210>3

<211>26

<212>DNA

<213> Artificial sequence

<400>3

GCGAATTCAT TCCGGTGCGT CGATCG 26

<210>4

<211>30

<212>DNA

<213> Artificial sequence

<400>4

CGCTCGAGTT ATTCCCAGATGACCACATCT 30

Claims (6)

1. a recombinant carrier of corn smut effector protein Pit2 gene, is characterized in that: the preparation method of described recombinant carrier is as follows: extract corn RNA, and reverse transcription goes out cDNA; Take cDNA as template amplification Pit2 gene; The amplified product of Pit2 was digested by BamHI and XhoI, and inserted into the pET-28a expression vector by DNA ligase to obtain the recombinant plasmid pET-28a-Pit2. 2. the recombinant vector according to claim 2, is characterized in that: take cDNA as template, utilize following primer to amplify Pit2 gene, and primer sequence is as follows: Upstream primer: Pit2-F: GC GAATTC ATTCCGGTGCGTCGATCG underlined is the BamHI restriction site; Downstream primer: Pit2-R: CG CTCGAG TTATTCCCAGATGACCACATCT underlined is the XhoI restriction site; The upstream and downstream primers are shown in SEQ ID NO.3 and 4 respectively. 3. a method for expressing corn smut effector protein Pit2 gene, is characterized in that, comprises the following steps: (1) Transform Escherichia coli cells with the recombinant vector described in claim 1 or 2 to obtain a recombinant prokaryotic expression strain expressing Pit2; (2) Inoculate the recombinant prokaryotic expression strain into kanamycin liquid medium or kanamycin+chloramphenicol liquid medium, and cultivate the activated strain overnight; (3) The activated recombinant prokaryotic expression strain was transferred to the kanamycin liquid medium, and IPTG was added after shaking culture to induce the expression of recombinant Pit2; (4) After the induction is completed, the expressed Pit2 recombinant protein is recovered and purified. 4. the method for expressing maize smut effector protein Pit2 gene according to claim 3, is characterized in that, in step (3), when shaking culture to OD600=0.5, adding the IPTG that final concentration is 1.0mM, at 25 ℃ induction culture. 5. the method for expressing corn smut effector protein Pit2 gene according to claim 3, is characterized in that, kanamycin is 50ug/mL in the kanamycin liquid culture medium. 6. the method for expressing corn smut effector protein Pit2 gene according to claim 3, is characterized in that, kanamycin is 50ug/mL in described kanamycin+chloramphenicol liquid medium, and chloramphenicol The element is 50ug/mL.