CN104673822A - Recombinant vector and its preparation method and use - Google Patents

Abstract

The invention provides a recombinant vector and its preparation method and use. The recombinant vector is constructed from a yeast secretory expression vector pPICZaA, contains a human-derived CTLA4 extracellular region gene for expression of a human-derived CTLA4 fusion protein, also contains a Kex2 protease cleavage site, His label and c-myc label, can release the expressed CTLA4 fusion protein in a secretory protein form out of cells, realizes purification of the protein, and provides a simple method for CTLA4 protein industrialization.

Description

A kind of recombinant vector and its preparation method and application

technical field

The invention relates to the field of biotechnology, in particular to a recombinant vector and its preparation method and application.

Background technique

Cytotoxic T lymphocyte-associated antigen 4 (CTLA4) is a receptor on the surface of T lymphocytes and a co-stimulatory molecule that inhibits T cell activation. When the T cell surface receptor CTLA4 binds to the B7 molecule on the APC, it will inhibit T cells. Activation, thereby inhibiting the hyperactivation of T cells. Many diseases caused by excessive immune response are closely related to the low expression of CTLA4 protein, such as immune rejection caused by allogeneic transplantation, chronic bronchial asthma, rheumatoid arthritis (RA), AIDS, systemic lupus erythematosus, silver Psoriasis, endotoxic shock disease and other diseases, so CTLA4 protein or fusion protein has very broad application prospects in the specific treatment of these diseases. In addition, the overexpression of CTLA4 protein in the body is closely related to many diseases, especially tumors, such as melanoma, leukemia, some gastric and esophageal cancers, and Leishmaniasis. CTLA4 mAb, a monoclonal antibody to CTLA4, can be widely used in anti-tumor immunotherapy, and is currently a research hotspot and direction of tumor gene therapy. Obtaining recombinant proteins with biological functions and close to human natural CTLA4 protein is the key to CTLA4-specific treatment of diseases.

Therefore, it is necessary to provide an expression vector for expressing CTLA4 recombinant protein in eukaryotic cells.

Contents of the invention

In order to solve the above problems, the first aspect of the present invention provides a recombinant vector, the recombinant vector is a yeast secretion expression vector pPICZaA recombinant vector, the recombinant vector contains human CTLA4 extracellular region gene, can be used to express human CTLA4 fusion protein. The recombinant vector also contains Kex2 protease cleavage site, His tag and c-myc tag, which can make the expressed CTLA4 fusion protein released into the extracellular space in the form of secreted protein, and the protein can be purified to provide a good source for the industrial production of CTLA4 protein. a simple method. The second aspect of the present invention provides a recombinant bacterium comprising the recombinant vector described in the first aspect. The third aspect of the present invention provides a preparation method of the recombinant vector. The fourth aspect of the present invention provides a gene, which is a DNA molecule with a nucleotide sequence as shown in SEQ ID NO:1. The fifth aspect of the present invention provides an application of a recombinant vector.

In a first aspect, the present invention provides a recombinant vector comprising the gene shown in SEQ ID NO: 1, the recombinant vector is a recombinant pPICZaA plasmid, and the gene is inserted into the multiple cloning site of the pPICZaA plasmid.

The pPICZaA plasmid used in the present invention contains His tag (histidine tag) and c-myc tag, which can make the expressed protein carry these two tags. The His tag is beneficial to the separation and purification of the fusion protein after expression, and the c-myc tag It is conducive to the analysis and tracking of fusion proteins in experiments, such as the analysis when used in western blot experiments.

Preferably, the gene as described in the first aspect is inserted into the Xho I and Xba I sites of the pPICZaA plasmid.

In a second aspect, the present invention provides a recombinant bacterium comprising the recombinant vector as described in the first aspect, the recombinant bacterium is a recombinant Pichia pastoris GS115 strain.

The nucleotide sequence shown in SEQ ID NO: 1 provided by the present invention includes the coding sequence of Kex2 protease cleavage site and the coding sequence of CTLA4 extracellular region (Gene Bank clone number: NM-005214.3).

Kex2 protease is located in the yeast cell membrane and is a cleavage enzyme for the α-factor signal peptide. It can effectively recognize the enzyme cleavage site Lys-Arg, and obtain the natural N-terminal CTLA4 protein by cleavage of the signal peptide before the enzyme cleavage site (due to The upstream of the Kex2 restriction site provided by the present invention is the Xho I site of the plasmid, and the downstream is adjacent to the CTLA4 protein extracellular region sequence. Therefore, the natural N-terminal CTLA4 protein extracellular region can be obtained after cutting; the natural N-terminal refers to does not contain any other redundant series at the N-terminus of the extracellular domain of CTLA4 protein).

After the nucleotide sequence shown in SEQ ID NO:1 provided by the present invention is expressed in yeast, a CTLA4 extracellular region fusion protein containing a Kex2 protease cleavage site can be obtained, and the Kex2 protease cleavage site passes through the yeast cell membrane The Kex2 protease cleavage promotes the secretion of CTLA4 extracellular domain fusion protein to the extracellular space.

In a third aspect, the present invention provides a method for preparing a recombinant vector as described in the second aspect, comprising the following steps:

(1) Designing upstream and downstream primers for the extracellular region of the CTLA4 gene, the base sequences of the upstream and downstream primers are shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively;

(2) Provide or prepare a CTLA4 gene template, and use the upstream and downstream primers obtained in step (1) as PCR primers to amplify the fragment of the extracellular region of the CTLA4 gene;

(3) Take the pPICZaA plasmid, carry out a double enzyme digestion reaction with the CTLA4 gene extracellular region fragment amplified in step (2) and the pPICZaA plasmid respectively with the same endonuclease, purify and recover, and connect to obtain the recombinant vector.

Preferably, in the step (2), the CTLA4 gene template is a recombinant vector pGEM-T-CTLA4 containing the CTLA4 gene.

The pGEM-T-CTLA4 plasmid used in the present invention contains the full-length ORF of human CTLA4 gene (GeneBank clone number is NM-005214.3)

Preferably, in the step (3), the endonucleases used in the double digestion reaction are Xho I endonuclease and Xba I endonuclease.

The upstream primer shown in SEQ ID NO:2 is specifically:

5'-CCGCTCGAGAAAAGAAAAGCAATGCACGTGGCC-3'

The downstream primer shown in SEQ ID NO:3 is specifically:

5'-GCTCTAGAGCGTCAGAATCTGGGCACGGTT-3'

The composition of the upstream primer (CTLA4up) and downstream primer (CTLA4down) is as follows:

In a fourth aspect, the present invention provides a gene, which is a DNA molecule with a nucleotide sequence as shown in SEQ ID NO:1.

In the fifth aspect, the present invention provides the application of the recombinant vector according to the second aspect or the preparation method of the recombinant vector according to the fourth aspect in the preparation of drugs for immune or tumor diseases.

The recombinant vector constructed in the present invention can be used to express the extracellular region of human CTLA4 protein, which is encoded by the full-length gene sequence of the extracellular region of human CTLA4 protein. The pPICZaA-CTLA4 recombinant vector constructed in the present invention and the yeast strain GS115/pPICZaA- CTLA4 can be widely used in biopharmaceuticals, antibody drugs, protein production and other fields.

The recombinant vector provided by the present invention and its preparation method and application have the following beneficial effects:

(1) The recombinant vector provided by the present invention adopts the pPICZaA plasmid, and the gene encoding the Kex2 protease cleavage site and the gene containing the extracellular domain of human CTLA4 are constructed into the expression vector, which can be used to express the secreted human CTLA4 fusion protein.

(2) Secondly, the recombinant vector also has a His tag and a c-myc tag, which is helpful for protein purification and analysis;

(3) In addition, the preparation method of the recombinant vector provided by the present invention is simple and easy for industrial production.

Description of drawings

Fig. 1 is the construction flow chart of pPICZaA-CTLA4 recombinant vector provided by the present invention;

Fig. 2 is the agarose gel electrophoresis figure of the Kex2-CTLA4 gene that the embodiment of the present invention makes;

Fig. 3 is the plasmid map of the recombinant vector prepared in the embodiment of the present invention.

Detailed ways

The following description is a preferred embodiment of the present invention, it should be pointed out that for those skilled in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications are also considered Be the protection scope of the present invention.

The plasmid vectors pGEM-T-CTLA4 and pPICZaA plasmids used in the examples of the present invention, and the yeast strain GS115 used are commercially available, and the reagents used are all commercially available.

Combining with the pPICZaA-CTLA4 recombinant vector construction flow chart shown in Figure 1, the embodiment of the present invention provides a preparation method of the recombinant vector, comprising the following steps:

(1) Cloning of Kex2-CTLA4 fusion gene

a) providing an upstream primer and a downstream primer, wherein the base sequence of the upstream primer is shown in SEQ ID NO: 2, and the base sequence of the downstream primer is shown in SEQ ID NO: 3;

b) Provide the pGEM-T-CTLA4 plasmid as a DNA template, use PCR to amplify the CTLA4 extracellular region gene sequence, and configure the amplification system as follows, the primers in the system use the upstream primers and downstream primers described in step (1):

PCR reaction system

The PCR amplification program was: pre-denaturation at 94°C for 2 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 40 s, and after 30 cycles, extension at 72°C for 10 min. Take 5 μL of the reaction product and use 1.5% agarose gel electrophoresis to identify it. After the identification is correct, the PCR product is recovered from the gel.

(2) Construction of recombinant vector pPICZaA-CTLA4 containing Kex2-CTLA4 fusion gene

1. Double digestion of plasmid containing Kex2-CTLA4 fusion gene and pPICZaA;

Take the pPICZaA plasmid, and use the same enzymes to carry out the digestion reaction of the gene fragment amplified in step (1) and the pPICZaA plasmid respectively. The digestion systems are as follows:

2. Connection of Kex2-CTLA4 fusion gene and pPICZaA;

After performing 1% agarose gel electrophoresis on the double-digested product obtained in step 1, the target fragment was recovered, and the Kex2-CTLA4 fusion gene was ligated with pPICZaA at 22°C for 5 hours. The ligation reaction system was as follows:

3. Enzyme digestion and sequencing identification

The ligation product was transformed into DH5α competent cells, spread on LB solid plate medium containing Amp, cultured overnight at 37°C, picked a single colony and cultured, extracted the plasmid and identified it by enzyme digestion. The enzyme digestion system is as follows:

Enzyme digested overnight at 37°C, identified by 1% agarose gel electrophoresis, the agarose gel electrophoresis after digestion is shown in Figure 1, in Figure 2, lane M is the DNA molecular weight marker (1Kb Plus DNA Ladder, invitrogen ), Lane 1 is the result of double enzyme digestion (Xho Ⅰ and Xba Ⅰ) of the constructed recombinant vector pPICZaA-CTLA4, wherein, Lane 1 has an obvious band at about 400bp, and the size of this band is the size of the Kex2-CTLA4 fusion gene ( about 400bp), indicating that the Kex2-CTLA4 fusion gene was successfully constructed on the vector;

Sequence the identified positive clones, compare the inserted nucleotide fragment with the NCBI human CTLA4 fragment, and save the vector with the same base sequence. The plasmid map of the recombinant vector pPICZaA-CTLA4 (3898bp) provided by the present invention As shown in Figure 3.

Claims (8)

1. a recombinant vectors, is characterized in that, comprises the gene as shown in SEQ ID NO:1, and described recombinant vectors is restructuring pPICZaA plasmid, and described gene inserts the multiple clone site of described pPICZaA plasmid.

2. a recombinant bacterium, is characterized in that, comprises recombinant vectors described in claim 1.

3. recombinant bacterium according to claim 2, is characterized in that, described recombinant bacterium is restructuring Pichia pastoris GS115 bacterial strain.

4. a preparation method for recombinant vectors as claimed in claim 1, is characterized in that, comprises the steps:

(1), the upstream primer of design CTLA4 gene extracellular domain fragment and downstream primer, the base sequence of described upstream primer and downstream primer is respectively as shown in SEQ ID NO:2 and SEQ ID NO:3;

(2), provide or prepare CTLA4 gene template, and with step (1) gained upstream primer and downstream primer for PCR primer, amplification CTLA4 gene extracellular domain fragment;

(3), get pPICZaA plasmid, step (2) increased the CTLA4 gene extracellular domain fragment obtained and described pPICZaA plasmid utilize identical restriction endonuclease to carry out double digestion reaction respectively, and purifying connects after reclaiming, and obtains described recombinant vectors.

5. the preparation method of a kind of recombinant vectors as claimed in claim 4, is characterized in that, in described step (2), described CTLA4 gene template is the recombinant vectors pGEM-T-CTLA4 containing CTLA4 gene.

6. the preparation method of a kind of recombinant vectors as claimed in claim 4, is characterized in that, in described step (3), it is Xho I restriction endonuclease and Xba I restriction endonuclease that described double digestion reacts the restriction endonuclease adopted.

7. a gene, is characterized in that, described gene is the DNA molecular of nucleotide sequence as shown in SEQ ID NO:1.

8. the application of recombinant vectors as claimed in claim 1 in preparation immunity or tumor disease medicine.