Cordyceps militaris agglutinin gene and preparation method thereof, recombinant expression vector, strain, and preparation method and application of cordyceps militaris agglutinin
阅读说明：本技术 蛹虫草凝集素基因及其制备方法、重组表达载体、菌株、及蛹虫草凝集素的制备方法和应用 (Cordyceps militaris agglutinin gene and preparation method thereof, recombinant expression vector, strain, and preparation method and application of cordyceps militaris agglutinin ) 是由 杨庆 宫智勇 许琳 于 2020-07-31 设计创作，主要内容包括：本发明公开一种蛹虫草凝集素基因及其制备方法、重组表达载体、菌株、及蛹虫草凝集素的制备方法和应用,其中,所述蛹虫草凝集素基因CMA用于编码蛹虫草凝集素,所述蛹虫草凝集素基因CMA的核苷酸序列如SEQ ID NO：1所示。本发明提供的蛹虫草凝集素基因CMA,为从蛹虫草基因组中克隆出的部分基因片段,并确定了其为蛹虫草凝集素编码基因；此外,该蛹虫草凝集素基因CMA可以编码出一种蛹虫草凝集素,该蛹虫草凝集素能特异性凝集大鼠的红细胞,还对糖结构的识别和结合具有高度特异性,同时能特异性的识别、结合并激活巨噬细胞。(The invention discloses a cordyceps militaris agglutinin gene and a preparation method thereof, a recombinant expression vector, a strain, a preparation method and application of cordyceps militaris agglutinin, wherein the cordyceps militaris agglutinin gene CMA is used for coding cordyceps militaris agglutinin, and the nucleotide sequence of the cordyceps militaris agglutinin gene CMA is shown as SEQ ID NO: 1 is shown. The Cordyceps militaris agglutinin gene CMA provided by the invention is a partial gene fragment cloned from a Cordyceps militaris genome, and is determined to be a Cordyceps militaris agglutinin coding gene; in addition, the cordyceps militaris agglutinin gene CMA can encode cordyceps militaris agglutinin which can specifically agglutinate red blood cells of rats, has high specificity on the recognition and combination of sugar structures and can specifically recognize, combine and activate macrophages.)
1. A Cordyceps militaris agglutinin gene CMA is used for coding Cordyceps militaris agglutinin and is characterized in that the nucleotide sequence of the Cordyceps militaris agglutinin gene CMA is shown as SEQ ID NO: 1 is shown.
2. The preparation method of the cordyceps militaris lectin gene CMA as claimed in claim 1, comprising the following steps:
extracting total RNA from cordyceps militaris;
carrying out reverse transcription on the extracted total RNA to obtain cDNA;
performing PCR amplification by using the cDNA as a template and a specific primer pair to obtain a cordyceps militaris agglutinin gene CMA;
wherein the specific primers comprise any one of a first primer pair and a second primer pair, the first primer pair comprises a forward primer PF1 and a reverse primer PR1, and the second primer pair comprises a forward primer PF2 and a reverse primer PR 2;
the nucleotide sequence of the first primer pair is as follows:
forward primer PF 1: 5- 'ATGGAGATCGCAGAACAGAACGTC' -3 of the formula,
reverse primer PR 1: 5- 'TTACACGGTAATGATCTTATAGTCGCC' -3;
the nucleotide sequence of the second primer pair is as follows:
forward primer PF 2: 5- 'ATGGAGATCGCAGAACAGAACGTCGGT' -3 of the formula,
reverse primer PR 2: 5- 'TTACACGGTAATGATCTTATAGTCGCCTT' -3.
3. A recombinant expression vector comprising the Cordyceps militaris lectin gene CMA of claim 1.
4. The recombinant expression vector of claim 3, wherein the expression vector of the recombinant expression vector is plasmid pGEX-6P-1.
5. A strain comprising the Cordyceps militaris lectin gene CMA of claim 1.
6. The strain of claim 5, wherein the host cell of the strain is BL21(DE 3).
7. A preparation method of Cordyceps militaris agglutinin is characterized by culturing the strain of claim 5 or 6 to obtain Cordyceps militaris agglutinin.
8. Use of the Cordyceps militaris lectin of claim 1 in the preparation of a specific macrophage activating reagent.
9. The use of the Cordyceps militaris lectin as defined in claim 1 in the preparation of a reagent for specifically agglutinating rat erythrocytes.
10. Use of the Cordyceps militaris lectin as defined in claim 1 in the preparation of a reagent that specifically recognizes and binds carbohydrate structures.
Technical Field
The invention relates to the technical field of genetic engineering, and particularly relates to a cordyceps militaris agglutinin gene CMA and a preparation method thereof, as well as a recombinant expression vector, a recombinant expression strain, a preparation method and application of cordyceps militaris agglutinin.
Background
Lectins are a class of non-immunogenic globulins that selectively recognize and bind to specific carbohydrate structures. Lectins are widely distributed in nature and their presence is identified in bacteria, fungi, algae, mammals and plants. Macrofungi are a precious resource pool of lectins, and many fungi-derived lectins are reported to have high specificity and selectivity for sugar recognition and binding, and have been widely used in sugar biology research. On the other hand, the fungal lectins have a wide range of biological activities, such as antibacterial, antitumor, and immunomodulatory, and are receiving attention from researchers as an important class of potential agents/drugs.
Cordyceps militaris belongs to an important large fungus for both food and medicine, and contains polypeptide, polysaccharide, cordycepin, cordycepic acid and other active ingredients. The active ingredients endow the cordyceps militaris with important pharmacological activities, such as antibiosis, inflammation inhibition, immunity regulation, tumor resistance and the like. However, few studies on the characteristics and functions of the lectin gene of cordyceps militaris are currently performed.
Disclosure of Invention
The invention mainly aims to provide a cordyceps militaris agglutinin gene and a preparation method thereof, a recombinant expression vector, a strain, a preparation method and application of the cordyceps militaris agglutinin, and aims to provide the cordyceps militaris agglutinin gene which can encode the cordyceps militaris agglutinin with a specific function.
In order to realize the purpose, the invention provides a cordyceps militaris agglutinin gene CMA for coding cordyceps militaris agglutinin, wherein the nucleotide sequence of the cordyceps militaris agglutinin gene CMA is shown as SEQ ID NO: 1 is shown.
The invention also provides a preparation method of the cordyceps militaris agglutinin gene CMA, which comprises the following steps:
extracting total RNA from cordyceps militaris;
carrying out reverse transcription on the extracted total RNA to obtain cDNA;
performing PCR amplification by using the cDNA as a template and a specific primer pair to obtain a cordyceps militaris agglutinin gene CMA;
wherein the specific primers comprise any one of a first primer pair and a second primer pair, the first primer pair comprises a forward primer PF1 and a reverse primer PR1, and the second primer pair comprises a forward primer PF2 and a reverse primer PR 2;
the nucleotide sequence of the first primer pair is as follows:
forward primer PF 1: 5- 'ATGGAGATCGCAGAACAGAACGTC' -3 of the formula,
reverse primer PR 1: 5- 'TTACACGGTAATGATCTTATAGTCGCC' -3;
the nucleotide sequence of the second primer pair is as follows:
forward primer PF 2: 5- 'ATGGAGATCGCAGAACAGAACGTCGGT' -3 of the formula,
reverse primer PR 2: 5- 'TTACACGGTAATGATCTTATAGTCGCCTT' -3.
The invention also provides a recombinant expression vector which comprises the cordyceps militaris agglutinin gene CMA.
Optionally, the expression vector of the recombinant expression vector is plasmid pGEX-6P-1.
The invention also provides a bacterial strain, which comprises the cordyceps militaris agglutinin gene CMA.
Alternatively, the host cell of the strain is BL21(DE 3).
The invention also provides a preparation method of the cordyceps militaris agglutinin, which is used for culturing the strain to obtain the cordyceps militaris agglutinin.
The invention also provides application of the cordyceps militaris agglutinin in preparation of a specific macrophage activating reagent.
The invention also provides application of the cordyceps militaris agglutinin in preparation of a reagent for specifically agglutinating rat erythrocytes.
The invention also provides application of the cordyceps militaris agglutinin in preparation of a reagent for specifically recognizing and combining a sugar structure.
The Cordyceps militaris agglutinin gene CMA provided by the invention is a partial gene fragment cloned from a Cordyceps militaris genome, and is determined to be a Cordyceps militaris agglutinin coding gene; in addition, the cordyceps militaris agglutinin gene CMA can encode cordyceps militaris agglutinin which can specifically agglutinate red blood cells of rats, has high specificity on the recognition and combination of sugar structures and can specifically recognize, combine and activate macrophages.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other related drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is an electrophoresis diagram of the DNA encoding CMA in the preparation method of the Cordyceps militaris lectin gene CMA of the present invention after PCR amplification;
FIG. 2 is a SDS-PAGE test result chart of the expression of the Cordyceps militaris lectin gene CMA in the recombinant strain;
FIG. 3 is an elution pattern of Cordyceps militaris agglutinin CMA purified by glutathione affinity chromatography column;
FIG. 4 is a SDS-PAGE test result chart in the purification of Cordyceps militaris agglutinin CMA provided by the present invention;
FIG. 5 is a flow chart of the binding of Cordyceps militaris lectin to the surface of various cells;
FIG. 6 shows the effect of the Cordyceps militaris agglutinin provided by the present invention on macrophage M1 type and M2 type polarization factors under the condition of 0.25 μ M concentration;
FIG. 7 is the binding condition of the Cordyceps militaris lectin on the first 10 positions of the binding strength in the sugar chip;
FIG. 8 is a specific sugar chain structure of the sugar structure at the first 10 th position of the bonding strength provided in FIG. 7.
The implementation, functional features and advantages of the objects of the present invention will be further explained with reference to the accompanying drawings.
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.
The invention provides a cordyceps militaris agglutinin gene CMA for coding cordyceps militaris agglutinin, wherein the nucleotide sequence of the cordyceps militaris agglutinin gene CMA is shown as SEQ ID NO: 1 is shown. Specifically, the cDNAORF full length of the Cordyceps militaris agglutinin gene CMA is 411bp, 137 amino acids are coded in total, and the molecular weight is 15.3 kDa.
The Cordyceps militaris agglutinin gene CMA provided by the invention is a partial gene fragment cloned from a Cordyceps militaris genome, and is determined to be a Cordyceps militaris agglutinin coding gene; in addition, the cordyceps militaris agglutinin gene CMA can encode cordyceps militaris agglutinin which can specifically agglutinate red blood cells of rats, has high specificity on the recognition and combination of sugar structures and can specifically recognize, combine and activate macrophages.
The invention also provides a preparation method of the cordyceps militaris agglutinin gene CMA, which comprises the following steps:
step S10, extracting total RNA from Cordyceps militaris.
Specifically, Cordyceps militaris fruiting body (purchased from Guangzhou Yuexio edible fungus technologies, Inc.) is pulverized, and then Tiangen mirVana is usedTMAnd extracting the total RNA of the cordyceps militaris sporocarp by using the miRNA separation kit.
And step S20, carrying out reverse transcription on the extracted total RNA to obtain cDNA.
The total RNA extracted from the fruiting body of the cordyceps militaris is used as a template, and the reverse transcriptase and the primer are used for synthesizing the cDNA.
And step S30, carrying out PCR amplification by using the cDNA as a template and a specific primer pair to obtain the Cordyceps militaris agglutinin gene CMA.
Specifically, cDNA is taken as a template, and PCR amplification is carried out by using a specific primer pair to obtain a cordyceps militaris gene fragment, wherein the nucleotide sequence of the cordyceps militaris gene fragment is shown as SEQ ID NO: 1 and designated as CMA.
It should be noted that, the specific primer of the present invention is not specifically limited, as long as the cordyceps militaris lectin gene CMA can be obtained by PCR amplification. In the present invention, 2 specific examples of specific primers are proposed, namely, the specific primers comprise a first primer pair and a second primer pair, wherein the first primer pair comprises a forward primer PF1 and a reverse primer PR1, and the second primer pair comprises a forward primer PF2 and a reverse primer PR 2; the nucleotide sequence of the first primer pair is:
forward primer PF 1: 5- 'ATGGAGATCGCAGAACAGAACGTC' -3 of the formula,
reverse primer PR 1: 5- 'TTACACGGTAATGATCTTATAGTCGCC' -3;
the nucleotide sequence of the second primer pair is:
forward primer PF 2: 5- 'ATGGAGATCGCAGAACAGAACGTCGGT' -3 of the formula,
reverse primer PR 2: 5- 'TTACACGGTAATGATCTTATAGTCGCCTT' -3.
The invention also provides a recombinant expression vector which comprises the cordyceps militaris agglutinin gene CMA.
Expression vectors (Expression vectors) are vectors in which Expression elements (such as promoters, RBSs, terminators, and the like) are added on the basis of the basic skeleton of a cloning vector to enable a target gene to be expressed. In the present invention, the expression vector is plasmid pGEX-6P-1. Specifically, the Cordyceps militaris lectin gene CMA is inserted into an expression vector pGEX-6P-1 through enzyme digestion of Bam H1 and Xho1, and a recombinant expression vector can be obtained.
The invention also provides a bacterial strain, which comprises the cordyceps militaris agglutinin gene CMA.
The strain is a recombinant strain and can be obtained by transforming escherichia coli BL21(DE3) with a recombinant expression vector containing the cordyceps militaris agglutinin gene CMA. That is, the recombinant strain may further comprise the above-described recombinant expression vector. The recombinant expression vector is introduced into host cells to obtain recombinant strains, and the target gene can be replicated along with the propagation of the host cells. Generally speaking, the host cell may be Escherichia coli, or a cell such as yeast, or other cell types such as animal cells, and in this example, the expression cell is preferably Escherichia coli BL21(DE 3).
In view of this, the above recombinant strain can be prepared by the following steps:
step S1: the Cordyceps militaris agglutinin gene CMA is inserted into an expression vector pGEX-6P-1 through enzyme digestion of Bam H1 and Xho1 to obtain a recombinant expression vector;
step S2: and transforming the recombinant expression vector into escherichia coli BL21(DE3) to obtain recombinant escherichia coli BL21(DE3) containing the recombinant expression vector, namely the recombinant strain.
Based on the preparation method of the strain, the preparation method for culturing the strain to form the cordyceps militaris agglutinin can be obtained, and the preparation method can be realized by the following steps:
step A10, expressing the Cordyceps militaris agglutinin gene CMA fused with GST at the N end in the strain: inserting the N section of the obtained Cordyceps militaris agglutinin gene CMA into GST tag, inoculating Escherichia coli BL21(DE3) bacterial liquid of the Cordyceps militaris agglutinin gene CMA containing GST tag into liquid LB culture medium (volume ratio of ampicillin to LB culture medium is 1:1000) containing ampicillin at 1% (V/V), and culturing at 37 deg.C and 200rpm until OD6000.6-0.8, adding IPTG (isopropyl thiogalactoside) to a final concentration of 0.5mM, culturing at 18 ℃, 200rpm for 14-16 h, then centrifuging at 4 ℃, 4000-5000 rpm for 20min, collecting thalli sediment, adding PBS (phosphate buffer solution) to resuspend the thalli sediment, carrying out ultrasonic crushing (power is 200W, ultrasonic treatment is carried out for 10s, intermittent treatment is carried out for 10s, repeating for 90 times), then centrifuging at 10000rpm and 4 ℃ for 20min, and collecting supernatant;
step A20, purifying Cordyceps militaris agglutinin CMA: equilibrating the glutathione agarose column with 50mL of PBS buffer solution at a flow rate of 1mL/min, loading the supernatant collected in step A10 onto the glutathione agarose column at a rate of 500. mu.L/min, and loading with PBS buffer solutionWashing the column to remove non-specifically bound heteroproteins to A280 nmThe value is stable, and the target protein cordyceps militaris agglutinin CMA is eluted by using an elution buffer solution to obtain cordyceps militaris agglutinin;
wherein the pH value of the PBS buffer solution is 7.4, and the PBS buffer solution comprises the following components in molar concentration: 140mM NaCl, 2.7mM KCl, 10mM Na2HPO4·12H2O and 1.8mM KH2PO4(ii) a The PH of the elution buffer was 7.4, and the elution buffer included the following components in molar concentrations: 140mM NaCl, 2.7mM KCl, 10mM Na2HPO4·12H2O, 1.8mM KH2PO4And 10mM GSH.
In the above embodiments relating to gene cloning, preparation of recombinant expression vectors and recombinant strains, and obtaining of recombinant proteins by culturing recombinant strains, the specific experimental methods and experimental materials are not described, and they are performed according to the conventional methods for preparing proteins by genetic engineering and microbial fermentation culture based on genetic engineering.
The invention also provides application of the cordyceps militaris agglutinin in preparation of a specific macrophage activating reagent.
The Cordyceps militaris agglutinin gene CMA encodes Cordyceps militaris agglutinin, and is prepared by cloning partial gene fragment of Cordyceps militaris genome, wherein the gene fragment is Cordyceps militaris agglutinin gene CMA, introducing the Cordyceps militaris agglutinin gene CMA into Escherichia coli BL21(DE3) host cell to obtain recombinant strain, and culturing the recombinant strain. The Cordyceps militaris agglutinin can specifically identify, combine and activate macrophage M1 type polarization, and can be applied to preparation of specific macrophage activating reagent.
The invention also provides application of the cordyceps militaris agglutinin in preparation of a reagent for specifically agglutinating rat erythrocytes.
The invention also provides application of the cordyceps militaris agglutinin in preparation of a reagent for specifically recognizing and combining a sugar structure.
At present, no specific antibody exists in the study of the sugar structure, so the study is difficult. The cordyceps militaris lectin prepared by the invention can specifically identify and combine a sugar structure which takes core pentose as a framework and is subsequently connected with a plurality of Gal-GlcNAc-units, can be applied to the preparation of a reagent for specifically identifying and combining the sugar structure, and can also be applied to the research of a sugar chain structure.
The technical solutions of the present invention are further described in detail below with reference to specific examples and drawings, it should be understood that the following examples are merely illustrative of the present invention and are not intended to limit the present invention.