CN111733178A

CN111733178A - Recombinant expression vector for improving soluble expression quantity of disease course related protein of astragalus mongholicus

Info

Publication number: CN111733178A
Application number: CN202010667955.8A
Authority: CN
Inventors: 王珏
Original assignee: Shanxi University of Chinese Mediciine
Current assignee: Shanxi University of Chinese Mediciine
Priority date: 2020-07-13
Filing date: 2020-07-13
Publication date: 2020-10-02

Abstract

The invention discloses a recombinant expression vector for improving the soluble expression quantity of a disease course related protein of astragalus mongholicus, which takes pET30a as a vector and utilizes an escherichia coli molecular chaperone skp to modify a recon pET30a-skp-AmpR-10 constructed by AmpR-10. The invention improves the soluble expression quantity of the target protein by replacing the carrier and modifying the molecular chaperone, further realizes the purposes of saving cost and improving yield, and further performs activity determination on the exogenously expressed AmpR-10, which shows that the exogenously expressed protein also has nuclease activity, provides a referable strategy for the in vitro expression of other homologous related proteins, and the activity may have important significance for researching the capability of the astragalus to resist RNA viruses.

Description

Recombinant expression vector for improving soluble expression quantity of disease course related protein of astragalus mongholicus

Technical Field

The invention belongs to the field of genetic engineering, and particularly relates to a recombinant expression vector for improving the soluble expression quantity of a disease course related protein of astragalus mongholicus.

Background

Plant disease-related Proteins (PR) are produced when plants are invaded by pathogens and are subjected to external stress, and have important significance on the disease-resistant immunity of the plants. It is divided into 14 families, including PR-1 to PR-14. Studies have shown that the disease-related protein PR-10 is not only involved in the response of disease-resistant mechanisms in plants, but also plays a role in the development of plants themselves (Walter M H, Liu J W, Wunn J, et al. Bentorinucleic-lipid-like related protein genes (YPR-10) display compositions of developmental, dark-induced and exogenous proteins-dependent expension [ J ]. Eur J Biochem,1996,239: 281-293). In recent years, the disease course related protein AmPR-10 of Mongolian radix astragali has attracted attention, and researches prove that the natural AmPR-10 mature protein consists of 158 amino acids, has the theoretical size of 16.8KDa and has nuclease activity (Qilaugho, research on two bioactive proteins in Mongolian radix astragali [ D ], Chinese agriculture university, 2006; Renmhong, Xuehuiqing, Liu Yong, purification of the disease course related protein AmPR-10 of Mongolian radix astragali and research on biological functions, Chinese traditional medicine journal, 2018, 43: 3662-3666). It is also well documented that the nuclease activity of PR-10 type proteins may play a role in the defense of plants against pathogen infection and is associated with their resistance to RNA viruses (Wenyun, Haoweiren, Huangqun. disease course-associated protein 10 plays a role in plant defense responses, communicating with plant physiology). Therefore, the in vitro research on the performance of the AmPR-10 has important significance for further discussing the growth and development and disease resistance mechanism of the astragalus, but the traditional method for extracting the protein from the natural astragalus has higher cost, less protein yield and inconvenient subsequent research.

Disclosure of Invention

The purpose of the invention is as follows: in order to solve the technical problems in the prior art, the invention provides the recombinant expression vector for improving the soluble expression quantity of the disease course related protein of the astragalus mongholicus, which can effectively improve the soluble expression quantity of the target protein, thereby achieving the purposes of saving cost and improving yield.

In order to achieve the purpose, the technical scheme of the invention is as follows: the invention relates to a recombinant pET30a-skp-AmPR-10 constructed by modifying AmPR-10 by using a molecular chaperone skp of Escherichia coli and taking pET30a as a carrier, which is unexpectedly found to be capable of effectively improving the soluble expression quantity of a target protein.

The specific construction method comprises the following steps:

(1) obtaining a target gene AmPR-10 by using total RNA of Mongolian astragalus as a template through RT-PCR, wherein the target gene AmPR-10 consists of 158 amino acids, and the nucleotide sequence of the target gene AmPR-10 is shown as SEQ NO. 1; specifically, astragalus membranaceus leaves are taken, cut into pieces, weighed, ground into uniform slurry according to the volume of Trizol reagent required by weight calculation, extracted by chloroform, and centrifugally separated, and finally RNA is dissolved in a water phase. Then, PCR was performed using RNA as a template.

(2) Taking an escherichia coli k12 genome as a template, obtaining an escherichia coli molecular chaperone skp through PCR, wherein the escherichia coli molecular chaperone skp consists of 161 amino acids, and the nucleotide sequence of the escherichia coli molecular chaperone skp is shown in SEQ NO.2 (containing a terminator taa); this step can be accomplished by a bacterial genome extraction kit, extracting the genome of K12, followed by amplification of skp by PCR.

(3) The target gene is cloned by using an escherichia coli expression vector pET-30a, the skp is connected with a vector pET30a, and then the AmpR-10 is connected to the downstream of the skp to construct a recombinant pET30 a-skp-AmpR-10. The specific flow is shown in figure 1.

The invention further provides application of the recombinant expression vector in improving the soluble expression quantity of the disease course related protein AmPR-10 of the Mongolian astragalus.

Specifically, the recombinant pET30a-skp-AmPR-10 was cultured in LB liquid medium to a cell density OD₆₀₀Adding isopropyl thiogalactoside IPTG for induction when the temperature reaches 0.5-0.6, culturing at 37 deg.C for 4 hr respectively to induce protein expression, ultrasonic crushing to obtain crude protein solution of target protein, dissolving in Tris-HCl buffer solution, and passing through Ni²⁺And purifying the target protein by using the protein purification kit.

Specifically, IPTG was obtained at a final concentration of 0.1 mM.

Has the advantages that: the invention takes the AmpR-10 gene as a research object, establishes a soluble expression system of the gene in an escherichia coli body for the first time, improves the soluble expression quantity of target protein by replacing a carrier and a molecular chaperone modification method, further realizes the purposes of saving cost and improving yield, further performs activity determination on the AmpR-10 expressed by an exogenous source, not only proves that the protein expressed by the exogenous source has nuclease activity, but also provides a referable strategy for the in vitro expression of other homologous related proteins, and the activity possibly has important significance for researching the capability of astragalus to resist RNA viroid.

Drawings

FIG. 1 is a flow chart of the construction of the recombinant pET30 a-skp-AmPR-10;

FIG. 2 shows the amplification results of the bands of the genes AmPR-10 and skp;

FIG. 3 shows the results of recombinant identification, wherein (A) the result of recombinant pET28a-AmPR-10 identification; (B) the result of identifying the recon pET30 a-AmPR-10;

FIG. 4 shows the result of identifying the recombinant pET30 a-skp-AmPR-10;

FIG. 5 shows the protein expression and purification of the recombinant pET28a-AmPR10 at 37 ℃;

FIG. 6 shows the protein expression and purification of the recombinant pET30a-AmPR10 at 37 ℃;

FIG. 7 shows protein expression and purification of the recombinant pET30a-skp-AmPR10 at 37 ℃;

FIG. 8 shows the detection results of the target protease activity;

FIG. 9 shows the spatial conformational role changes of Val at position 39 and Thr at position 40 before and after S-tag modification;

FIG. 10 shows the conformational role changes at Asp position 91, Ala position 92 and Asn position 93 before and after modification of S-tag;

FIG. 11(A) is a simulation diagram of prediction of three-level structure of AmPR-10, and (B) is a simulation diagram of prediction of three-level structure of skp and AmPR-10 fusion expression.

Detailed Description

The present invention will be described in further detail with reference to specific examples. The examples will help to understand the present invention given the detailed embodiments and the specific operation procedures, but the scope of the present invention is not limited to the examples described below.

The invention takes the AmPR-10 gene as a research object, establishes a soluble expression system of the gene in an escherichia coli body, and improves the soluble expression quantity of target protein by a method of replacing a carrier and modifying molecular chaperones.

Strain material: mongolian milkvetch root leaves (planted by Shanxi university of traditional Chinese medicine); escherichia coli K12 is purchased from China general microbiological culture Collection center; coli BL21 was purchased from bio-engineering (shanghai) ltd.

Tool enzyme and vector: PCR polymerase, restriction enzymes NcoI, EcoRI, Xhol, T4DNA Rapid ligase were purchased from Biotechnology engineering (Shanghai) Ltd; the cloning expression vectors pET-28a and pET-30a are provided by the Experimental center of the basic medical college of Shanxi university of traditional Chinese medicine.

Kit and related reagents: bacterial genome extraction kit, RT-PCR kit, PCR Cleanup kit, plasmid extraction kit, gel purification kit and Ni²⁺Purified protein kits, yeast tRNAs, etc. were purchased from Biotechnology engineering (Shanghai) Ltd.

Example 1 recombinant construction.

(1) Extracting total RNA of Mongolian astragalus: 0.2g of Mongolian milkvetch root leaves are weighed, ground on ice until homogenate is obtained, and transferred to a centrifuge tube. 1ml Trizol reagent was added and left at room temperature for 5 min. Adding chloroform and isopropanol, centrifugally extracting, airing at room temperature, adding a buffer solution to dissolve RNA.

(2) Cloning a target gene: taking total RNA of Mongolian astragalus as a template, and obtaining a PCR product of AmPR-10 by using an RT-PCR kit, wherein the PCR product consists of 158 amino acids, and the protein size is 16.8 kD; extracting Escherichia coli K12 genome, and using it as template to obtain PCR product of Escherichia coli molecular chaperone skp, which is composed of 161 amino acids and has protein size of 17 kD. The target gene is cloned by using Escherichia coli expression vectors pET-28a and pET-30a, and 3 recombinants are constructed in total. The primers were designed using Primer software as shown in Table 1.

TABLE 1 primer sequence Table 1 for PCR of target gene

Escherichia coli molecular chaperone skp is obtained by PCR with genome of Escherichia coli k12 as template, and consists of 161 amino acids (NCBI: 000913.3), length is 483bp, band position is shown in figure 2 (

bands

1 and 2 are parallel samples), and the band position accords with expected size; the target gene AmPR-10 is obtained by RT-PCR with the total RNA of the leaves of the astragalus mongholicus as a template, and consists of 158 amino acids (NCBI: KY419098.1), the length is 474bp, the position of a strip is shown in figure 2 (

strips

3 and 4 are parallel samples), and the size is in accordance with the expected size.

2.1.2 recombinant identification

As shown in FIG. 3-A, the result of the identification of the recombinant pET28a-AmPR-10 is shown, the band 1 is an AmPR-10PCR band control, the band 2 is a plasmid pET28a band control, and the band 3 is an electrophoresis band of the recombinant after EcoRI and XhoI double digestion. Determining to obtain positive clones; as shown in FIG. 3-B, the result of the identification of the recombinant pET30a-AmPR-10 is shown, the band 1 is an AmPR-10PCR band control, the band 2 is a plasmid pET30a band control, and the band 3 is an electrophoresis band of the recombinant after EcoRI and XhoI double digestion. Determining to obtain positive clones;

as shown in FIG. 4, the target gene was amplified by PCR using the recombinant plasmid pET30a-skp-AmPR-10 as a template. The

bands

1 and 2 are parallel samples, and are the identification result of PCR of the whole gene consisting of skp and AmPR-10; the strip 3 is the result of single skp PCR identification; the strip 4 is a single AmPR-10PCR identification result; recombinants were successfully identified.

1.2.2 Induction purification of the protein of interest

Culturing the recombinant in LB liquid culture medium to the thallus concentration OD₆₀₀When the concentration reached 0.5-0.6, IPTG (IPTG final concentration reached 0.1mM) was added to induce protein expression, and the cells were cultured at 37 ℃ for 4 hours, respectively. Obtaining crude protein solution (dissolved in Tris-HCl buffer solution) of target protein by ultrasonic disruption, and then passing through Ni²⁺The protein purification kit purifies target protein, protein samples before and after purification are detected by SDS-PAGE, and correct expression is determined. Meanwhile, the concentration of the pure protein is determined by a Coomassie brilliant blue method and is used for detecting the subsequent activity.

1.2.3 detection of the Activity of the protein of interest

2mg of yeast tRNA was weighed and dissolved in 1ml of buffer (100 mmol. multidot.L-1 MES, pH 6.0), and 100. mu.l of the supernatant containing the target protein AmPR-10 was added thereto, followed by incubation at 50 ℃ for 30 min. Immediately adding 1mL of precooled lithium chloride to terminate the reaction after the reaction is finished, carrying out ice bath for 3h at 12000rpm at 4 ℃, centrifuging for 15min, taking supernate at 260nm, and measuring the supernateThe absorbance was measured by using a sample without the enzyme solution as a control. The enzyme activity is defined as 50 DEG C^[5]The amount of the enzyme which changed the A260 absorbance by 1.0 in 2ml of the reaction system under the reaction conditions of pH6.0 was defined as one activity unit U (defined by referring to the activity of a commercial enzyme Benzonase nuclease), and the specific activity (U/mg) of AmpR-10 was calculated from the purified enzyme concentration.

2.2.1 protein expression of pET28a-AmPR-10

FIG. 5-A shows the protein induction expression pattern of the recombinant pET28a-AmPR-10, and the band 1 is the electrophoresis band control of the non-induced supernatant sample of the recombinant; the band 2 is a recombinant non-induced inclusion body electrophoresis band control; the strip 3 is the expression condition of supernatant soluble protein induced by pET28a-AmPR-10 at 37 ℃; the band 4 is pET28a-AmPR-10, and the inclusion body expression is induced at 37 ℃. FIG. 5-B shows the target band of the purified soluble protein AmPR-10 with inducible expression, which is located at about 17 kD. As can be seen from the figure, the AmPR-10 is expressed correctly, but the soluble expression quantity is not optimistic, and most of the protein exists in the form of inclusion bodies.

2.2.2 protein expression of pET30a-AmPR10

As shown in FIG. 6-A, lane 1 is pET28a-AmPR-10 uninduced supernatant electrophoretic lane control; the strip 2 is the expression condition of soluble protein of a recombinant pET28a-AmPR-10 after being induced at 37 ℃; the strip 3 is the soluble protein expression condition of the recombinant pET30a-AmPR-10 after being induced at 37 ℃ (the expression quantity is obviously greater than that of the strip 2); the strip 4 is the expression condition of the inclusion body of the recombinant pET28a-AmPR-10 after being induced at 37 ℃, and compared with the strip 4 in the figure 6-A, the content is obviously reduced, which indicates that a part of the inclusion body exists in the form of soluble protein; strip 5 is pET30a-AmPR-10 uninduced supernatant electrophoretic strip control. FIG. 6-B shows the target band after soluble purification after induction of the recombinant pET30 a-AmPR-10.

2.2.3 protein expression of pET30a-skp-AmPR10

FIG. 7 shows the protein induction expression pattern of the recombinant pET30a-skp-AmPR-10, and the band 1 is the soluble protein expression of the recombinant after being induced at 37 ℃, compared with the band 3 in FIG. 6-A, the expression level is further increased, and the position of the protein band is about 34kD, which accords with the result of co-expression of the molecular chaperone skp and the disease course related protein AmPR-10. Band 2 is the corresponding purification result.

As shown in the above results, the target protein expressed by the three vectors (pET-28 a; pET30 a; pET30a-skp) was purified by the kit, and SDS-PAGE electrophoresis detected that the target band was correctly positioned, and then the specific activity of the target protein on catalysis of the yeast tRNA was calculated by measuring the concentration of the purified protein according to the definition of enzyme activity, and the results are shown in FIG. 8. As can be seen from the figure, the AmpR-10 genes constructed by the three vectors can all express nuclease activity through exogenous expression, and the average value is about 1.1U/mg, and the differences are not large.

3.1 Effect of S-tag on exogenous expression of proteins

According to the experiment, the result of comparing the exogenous expression quantity of the AmPR-10 gene recombined in three vectors is as follows: pET30a-skp-AmPR-10> pET30a-AmPR-10> pET28a-AmPR-10, wherein one of the structural differences of the vector pET30a and pET28a is that the former contains a label S-tag, the invention further carries out modeling analysis on the target protein before and after modification of the S-tag respectively (predicted address: http:// swissmodel. expasy. org /), and the conformation shown in figure 9 and figure 10 is obtained through a chimera software, and the modeling results indicate that the two have the following differences: 1. as shown in FIG. 9, after the AmPR-10 is modified by S-tag, two amino acids are added in the first section of alpha helical structure, namely Val and Thr, Val is hydrophobic amino acid, and Thr is polar amino acid, so that hydrogen bonds are easily formed, and the two amino acids enhance the stability of the alpha helix, thereby improving the overall stability of the protein, and therefore, the target protein modified by S-tag is more stable; 2. as shown in FIG. 10, Asp at position 91, Ala at position 92 and Asn at position 93 are linkers connecting two beta sheets (FIG. A), and after S-tag modification expression, the part forms a short alpha helix, and the alpha helix structure is shown in the literature to be beneficial to improving the stability of the protein [10], and Ala is a hydrophobic amino acid to be beneficial to enhancing the stability of the alpha helix [11 ]. Therefore, the expression amount of the recon pET30a-AmpR-10 to the AmpR-10 is higher than that of pET28a-AmpR-10 obtained by combining the analysis, and probably because the S-tag on the vector pET30a modifies and expresses the target protein, the proportion of the alpha helical structure is increased, so that the water delivery effect of the target protein is improved, the stability is enhanced, and the exogenous expression amount is finally improved.

3.2 Effect of chaperone skp on exogenous expression of proteins

FIG. 11(A) shows a graph of prediction of the tertiary structure of AmPR-10, which shows an alpha helix percentage of about 36.08% by SOPMA analysis; FIG. B is a diagram showing the prediction of the tertiary structure of the skp-modified fusion expression with the AmpR-10, in which part I indicates the tertiary structure of the AmpR-10, part II indicates the tertiary structure of the skp, the two parts are connected by a "linker", as shown by the dotted line in the figure, and the skp is bound to the N-terminal of the AmpR-10. The biological significance of chaperones is to help target proteins fold correctly, and in the natural host, eventually will be cut off and not become part of the mature protein. As shown in the graph (B), the simulation result conforms to the definition of molecular chaperone, the skp and the AmpR-10 are independently expressed, and the skp does not influence the spatial structure of the AmpR-10. Because the enzyme catalysis system for cutting off the molecular chaperone skp is deleted in an exogenous escherichia coli host, the two parts are co-expressed in a linker connection mode, and the molecular expression quantity is about 33kD according to SDS-PAGE detection. And the protein expression is improved once more (pET30a-skp-AmPR-10> pET30a-AmPR-10), and the consideration is that the molecular chaperone skp mainly consists of an alpha helical structure, so that the alpha helical proportion in the integral protein tertiary structure conformation formed by the molecular chaperone skp and the target protein AmPR-10 is improved, and according to the calculation and analysis of software, the alpha helical proportion can reach 78.87 percent, so that the hydrophobicity of the integral protein is improved, the protein spatial conformation is more stable, and the purpose of improving the expression level of soluble protein is realized.

The astragalus mongholicus course related protein AmPR-10 is successfully exogenously expressed in an escherichia coli body, the exogenously soluble expression quantity of the AmPR-10 is improved by replacing a carrier, biological functions of a carrier tag S-tag and a molecular chaperone skp are further discussed and determined by combining protein three-level structure simulation analysis, and the accumulation effect of the S-tag and the skp is utilized, so that the aim of obtaining a large amount of exogenously soluble AmPR-10 is fulfilled, the basis is made for further activity research of the exogenously related protein AmPR-10, and a referable strategy is provided for the in-vitro expression of other homologously related proteins.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention. All the components not specified in the present embodiment can be realized by the prior art.

Sequence listing

<110> Shanxi university of traditional Chinese medicine

<120> a recombinant expression vector for increasing the soluble expression level of a disease course-related protein of Astragalus mongholicus

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<210>1

<211>474

<212>DNA

<213> AmPR-10 Gene (Artificial Sequence)

<400>1

atgggtgtta taagtttcaa tgaagagacc atctcaactg tagctccagc taggctttac 60

aaagctctag ttacagattc tgacaccctc atcccaaaga ctattcctga gatccagagt 120

gttgaaattg ttgaaggaaa cggtggcccc ggaaccatca agaaactcac tttcgttgaa 180

gatggagaaa ccaagcatgt gttgcacaaa gtggaagtaa ttgatgatgc taacttggtg 240

tacaagtaca gcttagttgg aggtgtagga tttccagaga ctgtggagaa gatctcattt 300

gagggtaaat tggttgctgg tccagatgga ggatccattg caaaaatcac cgtgacatac 360

cacaccaaag gagatgctac tcccactgaa aaggaactct tagatggcaa agtgaagggt 420

gaggctctct tcaaagccct tgagggttac gttttggcca atcctgaata caag 474

<210>2

<211>486

<212>DNA

<213> chaperone skp (artificial sequence)

<400>2

gtgaaaaagt ggttattagc tgcaggtctc ggtttagcac tggcaacttc tgctcaggcg 60

gctgacaaaa ttgcaatcgt caacatgggc agcctgttcc agcaggtagc gcagaaaacc 120

ggtgtttcta acacgctgga aaatgagttc aaaggccgtg ccagcgaact gcagcgtatg 180

gaaaccgatc tgcaggctaa aatgaaaaag ctgcagtcca tgaaagcggg cagcgatcgc 240

actaagctgg aaaaagacgt gatggctcag cgccagactt ttgctcagaa agcgcaggct 300

tttgagcagg atcgcgcacg tcgttccaac gaagaacgcg gcaaactggt tactcgtatc 360

cagactgctg tgaaatccgt tgccaacagc caggatatcg atctggttgt tgatgcaaac 420

gccgttgctt acaacagcag cgatgtaaaa gacatcactg ccgacgtact gaaacaggtt 480

aaataa 486

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cgcgaattca tgggtgttat aagtttca 28

<210>4

<211>28

<212>DNA

<213> AmPR downstream primer (Artificial Sequence) of pET28a-AmPR-10

<400>4

cgcctcgagc ttgtattcag gattggcc 28

<210>5

<211>28

<212>DNA

<213> AmpR-10 upstream primer (Artificial Sequence) of pET30a-AmpR-10

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cgcgaattca tgggtgttat aagtttca 28

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cgcctcgagc ttgtattcag gattggcc 28

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<213> Skp downstream primer (Artificial Sequence) of pET30a-skp-AmpR-10

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gcggaattct ttaacctgtt tcagtacgtc 30

<210>8

<211>28

<212>DNA

<213> AmpR-10 upstream primer (Artificial Sequence) of pET30a-skp-AmpR-10

<400>8

cgcgaattca tgggtgttat aagtttca 28

<210>9

<211>28

<212>DNA

<213> AmPR-10 downstream primer (Artificial Sequence) of pET30a-skp-AmPR-10

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cgcctcgagc ttgtattcag gattggcc 28

Claims

1. A recombinant expression vector for improving the soluble expression quantity of a disease course related protein of Mongolian astragalus is characterized in that the recombinant expression vector takes pET30a as a vector and utilizes an escherichia coli molecular chaperone skp to modify a recon pET30a-skp-AmpR-10 constructed by AmpR-10.

2. The method of constructing a recombinant expression vector according to claim 1, comprising the steps of:

(1) obtaining a target gene AmPR-10 by using total RNA of Mongolian astragalus as a template through RT-PCR, wherein the target gene AmPR-10 consists of 158 amino acids, and the nucleotide sequence of the target gene AmPR-10 is shown as SEQ NO. 1;

(2) taking an escherichia coli k12 genome as a template, obtaining an escherichia coli molecular chaperone skp through PCR, wherein the escherichia coli molecular chaperone skp consists of 161 amino acids, and the nucleotide sequence of the escherichia coli molecular chaperone skp is shown in SEQ NO. 2;

(3) the target gene is cloned by using an escherichia coli expression vector pET-30a, and a recombinant pET30a-skp-AmpR-10 is constructed.

3. The use of the recombinant expression vector of claim 1 to increase the soluble expression level of the disease course associated protein AmPR-10 of astragalus mongholicus.

4. The use as claimed in claim 3, wherein the recombinant pET30a-skp-AmPR-10 is cultured in LB liquid medium to a cell concentration OD₆₀₀Adding IPTG (isopropyl thiogalactoside) for induction when the concentration reaches 0.5-0.6, respectively at 37^oC culturing for 4 hr to induce protein expression, ultrasonic crushing to obtain crude protein liquid of target protein, dissolving in Tris-HCl buffer solution, and passing through Ni²⁺And purifying the target protein by using the protein purification kit.

5. The use according to claim 4, wherein the final concentration of IPTG is 0.1 mM.