CN104593406A - PIRES/TgDHFR-TS eukaryotic expression recombinant plasmid as well as construction and application thereof - Google Patents

Abstract

The invention discloses a PIRES/TgDHFR-TS eukaryotic expression recombinant plasmid. The recombinant plasmid is constructed through the recombination of a toxoplasma dihydrofolate reductase-thymidylate synthase TgDHFR-TS gene and a plasmid vector pIRES, and the recombinant plasmid can be used for successfully expressing recombinant protein. The eukaryotic expression recombinant plasmid constructed by the invention can be used as a new vector, a to-be-researched toxoplasma gene is introduced to a multiple-cloning site of the vector and overexpression target gene stable tachyzoite clone screening is carried out with the addition of pyrimethamine, so as to provide a powerful tool support for the further study of the functions of the gene in toxoplasma.

Description

PIRES/TgDHFR-TS Eukaryotic expression recombinant plasmid and structure thereof and application

Technical field

The invention belongs to gene engineering technology field, specifically, be that toxoplasma gondii Tetrahydrofolate dehydrogenase-thymidylate synthase gene is incorporated in pIRES expression vector, build a kind of new Eukaryotic expression recombinant plasmid.The plasmid that the present invention builds can enter in toxoplasma tachyzoite by the method for transfection, carries out by adding Pyrimethamine hcl the screening that process LAN stablizes tachyzoite clone.

Background technology

Toxoplasma gondii ( toxoplasma gondii) be a kind of special sexual cell endoparasitism protozoon, the mankind and other warm-blooded animals can be infected, people's toxoplasma gondii infection in the whole world about 1/3.Toxoplasma gondii is generally inapparent infection, and patient is without obvious clinical symptom, but after its infection pregnant woman, often cause the Unusual pregnancy such as miscarriage, premature labor, stillbirth, deformity, in survivor, 80% has intelligent growth obstacle.The incidence of congenital toxoplasmosis in newborn infant is up to 1 ‰ ~ 6 ‰.In recent years, along with iatrogenic immunosuppression, the increasing of organ transplantation and AIDS patient, the sickness rate of toxoplasmosis has the trend of rising, even causes lethal injury.Therefore, the research of toxoplasma gondii Disease-causing gene function and mechanism of causing a disease thereof, contributes to the pathogenesis disclosing toxoplasma gondii, simultaneously for the control of toxoplasmosis provides new thinking.

The research of current gene or protein function adopts two kinds of strategies usually, the first builds the Eukaryotic expression recombinant plasmid of this gene, after its transfection host cell, filter out the cell clone (overexpression) of this gene of stably express, carry out gene or albumen Quality Research; It two is this gene struck from host cell low (knockdown) or knock out (knockout), then studies the function of this gene.

Pyrimethamine hcl (pyrimethamine) is one of key agents for the treatment of arch insect infection at present, its mechanism of action is the synthesis by hindering dihydrofolic acid, cause tetrahydrofolic acid (THFA) to generate to reduce, stop gondii nucleic acid synthesis, thus reach the effect suppressing toxoplasma gondii propagation.And in arc polypide process LAN Tetrahydrofolate dehydrogenase-thymidylate synthase gene (DHFR-TS) and coding protein, the dihydrofolic acid dyssynthesis because Pyrimethamine hcl effect causes can be compensated, continue its life cycle.

Summary of the invention

The object of this invention is to provide a kind of pIRES/ tgdHFR-TS Eukaryotic expression recombinant plasmid, and the construction process of this recombinant plasmid and application.

PIRES/ provided by the invention tgdHFR-TS Eukaryotic expression recombinant plasmid is by Tetrahydrofolate dehydrogenase-thymidylate synthase gene tgdHFR-TS fragment and expression vector pIRES recombinate and obtain, and have the nucleotide sequence shown in SEQ ID NO.4, described sequence amounts to 7973bp.The present invention introduces HA sequence label in described Eukaryotic expression recombinant plasmid.

The construction process of Eukaryotic expression recombinant plasmid of the present invention is the primer that design has nucleotide sequence shown in SEQ ID NO.1 and SEQ ID NO.2, is cloned obtain by polymerase chain reaction (PCR) method from the cDNA of toxoplasma tachyzoite tgdHFR-TS, and will xhoi and ecoRi double digestion tgdHFR-TS gene PCR product is connected to the B cloning site of the eukaryotic expression vector pIRES of same double digestion, builds the pIRES/ of restructuring tgdHFR-TS Eukaryotic expression recombinant plasmid.

Wherein, pIRES is a kind of single promotor dual-expression vector of mammalian cell, this carrier contains two multiple clone site (A and B), containing insertion point (IRES) in the rrna of encephalomyocarditis virus (ECMV) between two multiple clone site, high expression while allowing two bicistronic mRNA transcripts.Contain cytomegalovirus promoter in the upstream of multiple clone site A, efficiently can start two multiple clone site and introduce transcribing of gene.

The present invention is successful clone Tetrahydrofolate dehydrogenase-thymidylate synthase gene first tgdHFR-TS, and the multiple clone site B place this gene being introduced pIRES, construct Eukaryotic expression recombinant plasmid pIRES/ tgdHFR-TS.

Recombinant plasmid constructed by the present invention shows, on carrier through PCR, double digestion and sequence analysis tgdHFR-TS gene reading frame is complete, and direction of insertion is correct, and remains the HA label of carboxyl terminal, is convenient to the later stage judge by detecting HA label tgthe expression of DHFR-TS.

And then, the Eukaryotic expression recombinant plasmid pIRES/ that the present invention builds tgdHFR-TS as new carrier, can introduce the target gene needing research at its multiple clone site A place.Concrete, the target gene introduced is toxoplasma cdna, as genes such as SAG, ROP17, is convenient to the function studying this gene further after transfection toxoplasma tachyzoite.

By liposome-mediated transfection method, by pIRES/ tgdHFR-TS Eukaryotic expression recombinant plasmid transfecting eukaryotic cells, RT-PCR and Western blotting result shows, and this gene can at eukaryotic expression.

Pyrimethamine hcl effectively can suppress the propagation of toxoplasma gondii, introduces Eukaryotic expression recombinant plasmid of the present invention in toxoplasma tachyzoite, can the effect of antagonism Pyrimethamine hcl, makes tachyzoite continue propagation.Therefore, Eukaryotic expression recombinant plasmid pIRES/ of the present invention tgdHFR-TS can as a kind of new plasmid vector, introduce in tachyzoite by needing other genes of the toxoplasma gondii of research, the screening that process LAN gene of interest stablizes tachyzoite clone is carried out, for the function of further goal in research gene in toxoplasma gondii provides strong instrument support by adding Pyrimethamine hcl.

Accompanying drawing explanation

Fig. 1 is pIRES/ tgthe structure schematic diagram of DHFR-TS Eukaryotic expression recombinant plasmid.

Fig. 2 is the PCR primer of toxoplasma gondii DHFR-TS gene.

In figure, M:DNA molecule marker; 1: tgdHFR-TS RT-PCR product; 2: negative control.

Fig. 3 is pIRES/ tgthe bacterium colony PCR of DHFR-TS recombinant plasmid and double digestion qualification figure.

In figure, A:M:DNA molecule marker; 1,2,3,4:pIRES/ tgdHFR-TS bacterium liquid PCR primer; 5: negative control; B:M:DNA molecule marker; 1,2:pIRES/ tgdHFR-TS double digestion product.

Fig. 4 is that RT-PCR (A) and Western blotting (B) detect pIRES/ tgthe expression of DHFR-TS in HEK 293T cell.

In figure, A:M:DNA molecule marker; 1: the cell of transfection empty plasmid; 2: transfection pIRES/ tgthe cell of DHFR-TS; B:1: the cell of transfection empty plasmid; 2: transfection pIRES/ tgthe cell of DHFR-TS.

Embodiment

The structure material used in the embodiment of the present invention and the source of main agents.

Toxoplasma gondii RH strain: this laboratory is preserved, mouse peritoneal inoculation is gone down to posterity.Carrier for expression of eukaryon: pIRES, purchased from American Clontech company.HEK 293T cell, purchased from Beijing Union Medical College basis institute cell centre.Escherichia coli DH5 α, purchased from Beijing Quanshijin Biotechnology Co., Ltd.

Restriction enzyme xbai He noti, purchased from precious biotechnology (Dalian) company limited; Trizol, HiFi-MMLV cDNA first chain synthetic agent box, 2 × Es Taq MasterMix, T ₄dNA ligase, sepharose reclaim test kit, DL 2000 DNA Marker, the little extraction reagent kit of plasmid, little mouse-anti HA monoclonal antibody, little mouse-anti β-actin antibody, are century company limited purchased from Beijing health; Pre-dyed albumen Marker, purchased from Canadian Fermentas company; The goat anti-mouse IgG of horseradish enzyme labelling, purchased from mountain gold bridge company limited in Beijing; ECL luminescent solution, purchased from Beijing English lattice benefactor department.

Other reagent are domestic analytical pure.

Embodiment 1

1, reverse transcription PCR design of primers

According to toxoplasma gondii Tetrahydrofolate dehydrogenase-thymidylate synthase gene ( tgdHFR-TS) protein coding gene (gene accession number: L08489.1) designs primer, introduces restriction enzyme respectively at 5 ' end of upstream and downstream primer xbai He notidentification cutting sequence (underscore) of I, and before downstream primer terminator codon, introduce HA sequence label (double underline), be convenient to the expression detecting target protein subsequently through tag antibody, primer is synthesized by Sino-U.S. calm and peaceful (Beijing) company limited.

Upstream primer: 5 '-ctag tCTAGAaccgccatgcagaaaccggtgtgtctgg-3 '.

Downstream primer: 5 '-ATAAGAAT gCGGCCGCtTA tGCGTAGTCAGGTACGTCATATGGGT AgACAGCCATCTCCATCTGGATT-3 '.

2, Tetrahydrofolate dehydrogenase-thymidylate synthase gene tgthe pcr amplification of DHFR-TS

The inoculation of toxoplasma tachyzoite conventional mouse is gone down to posterity, and collects 5 × 10 ⁸after tachyzoite, extract tachyzoite total serum IgE by Trizol test kit specification sheets, ultraviolet spectrophotometer is quantitative, and get 2 μ g and carry out reverse transcription, all the other are stored in-80 DEG C.The synthesis reference reagent box specification sheets of cDNA first chain.

Take cDNA as template, with the primer PCR amplification of synthesis tgdHFR-TS gene.Reaction system is: 25mM MgCl ₂3.5 μ l, template 2 μ l, upstream and downstream primer (10 μMs/L) each 1 μ l, 2 × Es Taq MasterMix 25 μ l, adds deionized water to 50 μ l.Reaction conditions is: 94 DEG C of 5min; 94 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 90s, totally 35 circulations; 72 DEG C of 10min.

1% agarose gel electrophoresis detects amplified production, and agarose gel electrophoresis shows, and product fragment is special, and size is about 1860bp, and conform to theoretical value (Fig. 2).

3, Eukaryotic expression recombinant plasmid pIRES/ tgthe structure of DHFR-TS and qualification

At 37 DEG C, use restriction enzyme xbai He noti respectively double digestion pIRES carrier and tgdHFR-TS gene PCR amplified production, 1% agarose gel electrophoresis be separated enzyme cut after carrier and PCR primer, with glue reclaim test kit reclaim digestion products, the goal gene after being cut by enzyme and carrier are at T ₄under DNA ligase effect, 22 DEG C of reaction 15min.Connect product conversion competent cell DH5 α, the LB be spread evenly across containing penbritin is dull and stereotyped, 37 DEG C of incubated overnight.Picking mono-clonal is inoculated in the LB liquid medium containing penbritin (100 μ g/ml) and increases in a large number.

Get the LB nutrient solution that 4 pipe inoculations have mono-clonal bacterium colony respectively, first carry out bacterium liquid PCR positive clone identification, the PCR primer clip size of result display 1,2, No. 4 clone is about 1860bp, points out it to be pIRES/ tgdHFR-TS Positive recombinant clones (Fig. 3 A).

Extract the plasmid DNA of 1, No. 2 clone subsequently, carry out xbai He noti double digestion, obtains 6100bp and 1860bp two fragments (Fig. 3 B), consistent with expected results.

Embodiment 2

HEK 293T cell containing 10% new-born calf serum DMEM substratum in, in 37 DEG C, 5%CO ₂cultivate under saturated humidity, when Growth of Cells to 70 ~ 80% merges, according to Lipofectamine ^tM2000 specification sheetss, by liposome-mediated transfection method transfection Eukaryotic expression recombinant plasmid pIRES/ tgdHFR-TS.And the HEK 293T cell of transfection pIRES empty plasmid is as negative control.

Transfectional cell cultivates 72h, collecting cell, and extract total serum IgE and gross protein respectively, RT-PCR and Western blotting detects tgdHFR-TS gene and protein expression.

RT-PCR detects tgthe expression of DHFR-TS in HEK 293T cell, internal reference used is β-actin.

Upstream primer: 5 '-CCAAGGCCAACCGCGAGAAGATGAC-3 '.

Downstream primer: 5 '-AGGGTACATGGTGGTGCCGCCAGAC-3 '.

Annealing temperature 58 DEG C, concrete grammar is with above-mentioned tgthe amplification of DHFR-TS gene.

The extraction of total protein of cell adopts RIPA lysate, and protein quantification adopts BCA method.Get 30 μ g total proteins and carry out Western blotting detection tgthe expression of DHFR-TS in HEK 293T cell.Cell pyrolysis liquid is separated through SDS-PAGE, after transferring film and closed supervisor, with little mouse-anti HA monoclonal antibody (1: 1000) for primary antibodie, goat anti-mouse HRP-IgG (1: 5000) two resists, adopt ECL luminescent solution, X sheet exposes, development, fixing post analysis result.

Result shows, at transfection Eukaryotic expression recombinant plasmid pIRES/ tghave in the cell of DHFR-TS tgthe expression of DHFR-TS gene (Fig. 4 A) and albumen (Fig. 4 B), and the cell of transfection empty plasmid has no expression, detects in two groups of cells all have the expression of house-keeping gene β-actin (gene size is 592bp, molecular weight of albumen position 43KDa) simultaneously.

Claims (7)

1. a pIRES/ tgdHFR-TS Eukaryotic expression recombinant plasmid, has the nucleotide sequence shown in SEQ ID NO.4, by Tetrahydrofolate dehydrogenase-thymidylate synthase gene tgdHFR-TS fragment and plasmid vector pIRES recombinate and obtain.

2. Eukaryotic expression recombinant plasmid according to claim 1, is characterized in that introducing HA sequence label in described recombinant plasmid.

3. the construction process of Eukaryotic expression recombinant plasmid described in claim 1, is the primer that design has nucleotide sequence shown in SEQ ID NO.1 and SEQ ID NO.2, clones and obtain from toxoplasma tachyzoite cDNA tgdHFR-TS, and will xhoi and ecoRi double digestion tgdHFR-TS gene PCR product is connected to the B cloning site of the eukaryotic expression vector pIRES of same double digestion, builds the pIRES/ of restructuring tgdHFR-TS Eukaryotic expression recombinant plasmid.

4. Eukaryotic expression recombinant plasmid described in claim 1 is as the application of carrier for expression of eukaryon, it is characterized in that introducing target gene at its multiple clone site A.

5. application according to claim 4, is characterized in that the target gene of described introducing is toxoplasma cdna.

6. application according to claim 5, is characterized in that described toxoplasma cdna is ROP17 gene.

7. application according to claim 5, is characterized in that described toxoplasma cdna is SAG gene.