CN1304411C - Saltant dihydrofolic acid reductase gene and application in mammal cell gene expression thereof - Google Patents

Abstract

The invention discloses a nucleotide sequence and an amino acid sequence of a missense mutant dihydrofolate reductase capable of increasing the expression level of mammalian cells, and a method for expressing genes in mammalian cells by using the dihydrofolate reductase gene method.

Description

A mutant dihydrofolate reductase gene and its application in mammalian cell gene expression

field of invention

The invention belongs to the field of biotechnology, and describes the sequence of a missense mutant dihydrofolate reductase gene and the amino acid sequence encoded by the polynucleotide sequence, as well as the method of using the polynucleotide sequence and amino acid sequence to express in mammalian cells method.

Background technique

The expression vectors of mammalian cell expression systems generally contain selection markers that can screen out the integration of foreign genes or amplification systems that selectively increase the copy number. Dihydrofolate reductase is currently the most commonly used selective amplification system. Dihydrofolate reductase catalyzes the conversion of folic acid to tetrahydrofolate. Tetrahydrofolate is required for the biosynthesis of purines, thymidine monophosphate and glycine. Methotrexate can bind to dihydrofolate reductase and inhibit its activity leading to cell death. When cells containing the dihydrofolate reductase gene were grown in media with increasing concentrations of methotrexate, the gene for dihydrofolate reductase was simultaneously amplified. The gene copy number of dihydrofolate reductase in some cells that can resist high concentrations of methotrexate can reach thousands, and the expression of dihydrofolate reductase can be increased by thousands of times. Therefore, the pressurized selection of methotrexate to make dihydrofolate reductase and the target gene to be expressed co-amplify, thereby improving the expression level of the target gene is currently the most commonly used gene amplification method (Hansjrg Hauser et al. . Mammalian cell biotechnology in protein production. Berlin; New York: de Gruyter, 1997).

Since the dihydrofolate reductase amplification system selects and amplifies the target gene to be expressed through the inhibitory effect of methotrexate on dihydrofolate reductase, the activity and expression level of dihydrofolate reductase have a great influence on the engineering of screening. Cellular expression yields have a significant impact. The expression level of the mammalian expression system can be improved by using a weak promoter and intentionally damaging the conserved sequence of Kozak (Li Yuyang. Gene Expression Technology. Beijing: Science Press, 2001, 139-158). It is suggested that by intentionally reducing the activity and expression level of dihydrofolate reductase, it is helpful to screen high-expression cell lines to express the target gene.

Detailed description of the invention

The invention discloses a polynucleotide sequence and corresponding amino acid sequence of a missense mutant dihydrofolate reductase gene.

The missense mutated dihydrofolate reductase gene was obtained by PCR random mutation method, and the 77th amino acid of the encoded protein was changed from serine to threonine, and its full sequence is shown in SEQ ID NO2.

The changes in the protein structure of dihydrofolate reducing acid may be caused by changes in the amino acid sequence. The interleukin 10 expression vector constructed by using the mutated dihydrofolate reductase gene was transfected into CHO DHFR-cells to remove its dependent condition HT (hypoxanthine and thymidine mixture), and the mixed cells and single clones screened The expression and output of interleukin 10 in the cells were about 3 times higher than that of the wild type dihydrofolate reductase. It suggested that the 77th amino acid of dihydrofolate reductase changed from serine to threonine, which may reduce the activity of dihydrofolate reductase.

The invention discloses a method for expressing the gene encoding the missense mutant amino acid in mammalian cells. The method for expressing mammalian cells by the gene encoding the missense mutant amino acid provided in the present invention refers to using the gene encoding the missense mutant amino acid as a selection marker and amplification system to select transfected mammals cells, and methods of increasing expression levels in cells by methotrexate. The expression vector constructed by using this mutated dihydrofolate reductase gene can construct the target gene to be expressed and the mutated gene together on a carrier to transfect the cells, or construct them on different vectors respectively cells were co-transfected.

Description of drawings

The following drawings are used to illustrate specific embodiments of the invention, but not to limit the scope of the invention defined by the claims.

Figure 1 is a map of mutant gene screening expression plasmids constructed by the method of three-segment directional ligation. EF1α/HTLV is the promoter, hIL10 is interleukin 10, and IRES is the internal ribosome entry site. PCR refers to using the Diversify ^TM PCR Random Mutagenesis Kit. DNA fragments amplified by PCR with primers 1 and 2.

Figure 2 and Figure 3 are the plasmid maps for constructing expression plasmid A and expression plasmid B respectively by using primer 1 and primer 2 to amplify the wild-type dihydrofolate reductase gene and the mutated dihydrofolate reductase gene respectively .

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods that do not indicate specific conditions in the following examples are all according to conventional conditions such as: Sambrook etc., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's suggested conditions.

Example 1: Construction and screening of dihydrofolate reductase mutant gene

Dihydrofolate reductase mutant gene was constructed using Diversify ^TM PCR Random Mutagenesis Kit (Clonteeh Company). The PCR conditions are as follows: reaction volume, 50 μl; deionized water 31 μl, 10×TITANIUM Taq Buffer 5 μl, MnSO ₄ 4 μl, dGTP (2 mM) 5 μl, 50×DiversifydNTP Mix 1 μl, primer 1 (10 μM, see SEQ ID NO3) 1 μl, Primer 2 (10 μM, see SEQ ID NO4 for the sequence) 1 μl, dihydrofolate reductase gene template (1 ng/μl), TITANIUMTaq enzyme 1 μl; thermal cycle: 94°C, 30 seconds; 94°C for 30 seconds, 56°C for 30 seconds, 68 1 minute at °C, 30 cycles; 1 minute at 68 °C.

After PCR amplification, the amplified product of 610 bp was purified by tapping and recovered, digested with BamHI and EcoRI, and constructed an expression plasmid for mutant gene screening by using the method of three-segment directional ligation. The promoter of the screening expression plasmid is the EF-1α/eIF4g hybrid promoter, and the expression plasmid contains the white medium 10 gene, which is connected with the dihydrofolate reductase gene fragment to be screened through IRES. The above ligation reaction solution was transformed into DH5α cells, and the transformed bacteria solution was directly shaken to extract the plasmid.

The plasmid extracted above was transfected into CHO DHFR-cells with Lipofectamine (GIBCO company). After 48 hours of transfection, after the cells were confluent, they were passaged in six-well plates according to 1/4, 1/8 and 1/12, and the HT mixture was removed. liquid. The medium is α-MEM, plus 10% dialyzed serum (Hyclone Company).

After maintaining the culture for two weeks, use the method of paving agarose to pick the monoclonal cells to a 96-well plate. After the cells grow and confluence to 80-100% in the 96-well plate, take the culture medium and measure the expression concentration of interleukin 10 by ELISA . A total of 156 monoclonal cells were selected, and the expression level of the cell line with the highest expression level was 300ng/10 ⁶ cells·24 hours.

The chromosomal DNA of the cell line with the highest expression level was extracted by the method of phenol-chloroform, and conventional PCR amplification was carried out with primer 1 (see SEQ ID NO3 for the sequence) and primer 2 (see SEQ ID NO4 for the sequence), and the amplified product was tapped and purified Sequencing, it was found that the base of the dihydrofolate reductase gene at position +230 was converted from G to C, and the nucleotide sequence results are shown in SEQ ID NO1.

Example 2: CHO cells express interleukin-10

The wild-type dihydrofolate reductase gene and the screened mutated dihydrofolate reductase gene were respectively amplified by primer 1 and primer 2, and expression plasmids A and B were respectively constructed (refer to Example 1 for the construction method and plasmid map). Transfect CHO DHFR ⁻ according to the method in Embodiment 1, and detect the expression levels of interleukin 10 in mixed cells and picked monoclonal cells after removing HT. The interleukin-10 expression of the mixed cells was 36ng/10 ⁶ ·24 hours after the expression plasmid A was removed from the HT, and the expression of the interleukin-10 was 124ng/10 ⁶ ·24 hours after the expression plasmid B was removed from the HT. Pick 60 monoclonal cells expressing plasmids A and B respectively. The highest expression level of the monoclonal cell line expressing plasmid A is 108ng/10 ⁶ 24 hours, and the highest expression level of the monoclonal cell line expressing plasmid B is 380ng/10 ⁶ ·24 hours. It is suggested that the expression yield of the cells screened by the missense mutant dihydrofolate reductase gene obtained in the present invention is higher than that of the wild-type dihydrofolate reductase gene, and the expression yield of the target gene is increased by about 3 times.

Sequence Listing

(1) General information:

Invention name: A missense mutant dihydrofolate reductase and its coding sequence

Number of sequences: 4

(2) Information on SEQ ID NO1:

(1) sequence features:

(A) type: nucleic acid

(B) Length: 564 bases

(C) chain: double chain

(D) topology; linear

(II) Molecular type: cDNA

(III) Sequence description:

atggttcgaccattgaactgcatcgtcgccgtgtcccaagatatggggattggcaagaacggagacctacc

ctggcctccgctcaggaacgagttcaagtacttccaaagaatgaccacaacctcttcagtggaaggtaaac

agaatctggtgattatgggtaggaaaacctggttctccattcctgagaagaatcgacctttaaaggacagaat

taatatagttctcactagagaactcaaagaaccaccacgaggagctcattttcttgccaaaagtttggatgat

gccttaagacttattgaacaaccggarttggcaagtaaagtagagacatggtttggatagtcggaggcagttct

gtttaccaggaagccatgaatcaaccaggccacctcagactctttgtgacaaggatcatgcaggaatttgaa

agtgacacgtttttcccagaaattgatttggggaaatataaacttctcccagaatacccaggcgtcctctctga

ggtccaggaggaaaaaggcatcaagtataagtttgaagtctacgagaagaaagactaa

(3) Information on SEQ ID NO2:

(1) sequence features:

(A) Type: amino acid

(B) Length: 187 amino acids

(D) topology; linear

(II) Molecule Type: Protein

(III) Sequence description:

M V R P L N C I V A V S Q D M G I G K N G D L P W P P L R N E F K Y F Q R MT T T S S S V E G K Q N L V I M G R K T W F S I P E K N R P L K D R I N I V L T R E LK E P P R G A H F L A K S L D D A L R L I E Q P E L A S K V D M V W I V G G S S S VY Q E A M N Q P G H L R L F V T R I M Q E F E S D T F F P E I D L G K Y K L L P EY P G V L S E V Q E E K G I K Y K F E V Y E K K D

(4) Information on SEQ ID NO3

(1) sequence features:

(A) type: nucleic acid

(B) Length: 27 bases

(C) chain: single chain

(D) topology; linear

(II) Molecular type: oligonucleotide

(III) Sequence description:

ATTGGATCCATGGTTCGACCATTGAACT

(4) Information on SEQ ID NO4

(1) sequence features:

(A) type: nucleic acid

(B) Length: 29 bases

(C) chain: single chain

(D) topology; linear

(II) Molecular type: oligonucleotide

(III) Sequence description:

TCAGAATTCTGCATAGCTTTAGGAGGGGA

Claims (6)

1, a kind of dihydrofolate reductase gene of missense mutation is characterized in that it has the nucleotide sequence shown in the SEQ ID NO1.

2, a kind of Tetrahydrofolate dehydrogenase DHFR-S77T of missense mutation is characterized in that the 77th amino acids of Tetrahydrofolate dehydrogenase is converted to Threonine by Serine, and its amino acid complete sequence is seen SEQ ID NO2.

3, utilize the gene of aminoacid sequence in the coding claim 2 in host cell, to carry out the method for destination gene expression as selective marker and amplification system.

4, the method described in the claim 3, wherein said host cell are Chinese hamster ovary cell.

5, the encoding gene of the Tetrahydrofolate dehydrogenase DHFR-S77T of the missense mutation described in the claim 2 application in polypeptide and protein production as selective marker and amplification system.

6, the described application of claim 5, amplification system wherein are meant the raising that utilizes methotrexate concentration and the goal gene copy number are increased.

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