CN102352362A - Optimized danio rerio vitellogenin gene, and expression carrier and application thereof - Google Patents

Abstract

The invention discloses an optimized danio rerio vitellogenin (vtg) gene, and an expression carrier and an application thereof, which belong to the technical field of biological engineering. Danio rerio vtg 1 NCBI accession: NP_001038362 NP_739573 XP_691311 sequences are used as templates, escherichia coli optimization codons are used for replacing 19 escherichia coli rare codonss, 10 inside restriction sites of nucleic acid restriction enzymes such as NdeI, EcoliI, NotI, SacI, BamHI and the like are eliminated, new genes nvtg1 of the danio rerio vitellogenin VTG with complete codes are designed and synthetized, and the expression plasmid containing the genes nvtgl and the engineering bacteria containing the plasmid are constructed. The recombinant expression of the danio rerio vitellogenin VTG is realized for the first time, the VTG protein yield can reach 3mg/ml, Western experiments show that the antigenicity of the danio rerio vitellogenin VTG is obvious, the high-efficiency expression of the VTG protein in prokaryote escherichia coli is realized, and the gene is suitable for industrial production.

Description

Zebra fish egg yolk protein origin 1 gene and expression vector and the application optimized

Technical field

The present invention relates to the new gene nvtg1 of complete zebra fish (Danio rerio) the vitellogenin VTG of a kind of artificial reconstructed synthetic coding and according to the expression plasmid of its structure and contain the engineering bacteria of this expression plasmid, belong to field of genetic engineering.

Technical background

Oestrogenic hormon (Estrogen) can be divided into endogenous estrogen and exogenous oestrogenic hormon; Endogenous estrogen mainly refers to the hormone of human body ovary synthesis secretion; And exogenous oestrogenic hormon also is environmental estrogens; They can get in the organism, synthetic, the secretion through disturbing the organism endogenous estrogen, transhipment, combination, active reaction, metabolism, the effect of clearing up or producing similar organism endogenous estrogen.The environmental estrogens kind is a lot, mainly comprises synthetic oestrogenic hormon, plant estrogen, fungoid oestrogenic hormon, environmental chemical pollutants of hormone effect or the like is arranged.Environmental estrogens pollutes has become a serious environmental problem and social concern.

The foundation of the animal model of some research environment oestrogenic hormon eco-toxicities, like zebra fish (Danio rerio), Tang fish etc., research and monitoring that environmental estrogens is polluted have great importance.The research of signal path makes people recognize vitellogenin (Vitel logenin in the estrogenic body; VTG) etc. albumen is oestrogenic hormon and estrogen receptor (Estrogen receptors; ER) Tiao Kong downstream albumen, so VTG can be used as a kind of biomarker important, that can react estrogen level.

The biological intravital VTG protein content of testing environment oestrogenic hormon pollution mode needs the technology of effectively quantitative and qualitative examination; Its method of protein detection of specifically using is Western Blot and Elisa, and the precondition of the use of Western Blot and Elisa is to need VTG albumen mark article.At present, and the main natural VTG albumen from model animals of VTG albumen mark article (natural VTG, nVTG); But its natural content is few; Bring very big difficulty to separation and purification, (recombinant VTG rVTG) is elder generation and then effective means and use the genetically engineered recombinant technology to produce VTG.

Summary of the invention

A technical problem to be solved by this invention provides a kind of artificial reconstructed synthetic; The new gene nvtg1 of the zebra fish vitellogenin VTG that the coding that can in intestinal bacteria, efficiently express is complete; This artificial reconstructed synthetic nvtg1 gene has nucleotide sequence shown in the SEQ ID NO.1.

Said nucleotide sequence has used intestinal bacteria preferences codon.

The expression plasmid that uses said zebra fish vitellogenin VTG gene nvtg1 to make up belongs to protection scope of the present invention.

Said expression vector is an expression vector in the born of the same parents, is skeleton with pET-28a (+) preferably.

Use the transgenic cell line of said zebra fish vitellogenin VTG gene nvtg1 or expression vector establishment or the protection domain that genetic engineering bacterium all belongs to this patent.

The expression vector that another technical problem to be solved by this invention provides a kind of said zebra fish vitellogenin VTG gene nvtg1 or contains zebra fish vitellogenin VTG gene nvtg1 is in environment measuring, the application during food inspection, medical research and VTG antibody preparation are produced.

The present invention has designed and synthesized the new gene nvtg1 of coding zebra fish vitellogenin, has made up the engineering bacteria that is applicable to that the yellow proteinogen of fish-egg is expressed.Expression of results shows that the VTG protein yield can reach 3mg/ml, and the Western experiment shows that its antigenicity is remarkable, has realized VTG albumen high-efficient expression in the prokaryotic organism intestinal bacteria, is fit to suitability for industrialized production.

Description of drawings

Fig. 1 nvtg1/pET28a (+) physical map

The SDS-PAGE collection of illustrative plates of the recombinant expressed VTG of Fig. 2 and Western collection of illustrative plates

A. the SDS-PAGE of recombinant expressed rVTG

1. albumen Marker; 2. female zebra fish VTG sample; Deposition in the born of the same parents of (3.pET-28a+)/BL21 (DE3); 3. do not use the deposition of IPTG inductive nvtg1/pET28a (+)/BL21 (DE3); 4. do not use the deposition of IPTG inductive nvtg1/pET28a (+)/BL21 (DE3); The VTG albumen that the nVTG representative is natural; The VTG albumen that the rVTG representative is recombinant expressed.

B. the Western of recombinant expressed rVTG identifies

1. albumen Marker; 2. female zebra fish VTG sample; Deposition in the born of the same parents of (3.pET-28a+)/BL21 (DE3); 3. do not use the deposition of IPTG inductive nvtg1/pET28a (+)/BL21 (DE3); 4. do not use the deposition of IPTG inductive nvtg1/pET28a (+)/BL21 (DE3); The VTG albumen that the nVTG representative is natural; The VTG albumen that the rVTG representative is recombinant expressed.

Embodiment

Below come further to illustrate the present invention through embodiment, the following example is used for illustration purpose but not is used to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example is all operated according to the common described condition of molecular cloning handbook basically.

Embodiment 1 nvtg1 gene design

With the full gene of zebra fish (Danio rerio) vitellogenin vtg1 (Danio rerio; Vitellogenin 1; NCBIaccession:NP_001038362 NP_739573 XP_691311) sequence is initial preface; Utilize its 19 intestinal bacteria rare codons of intestinal bacteria optimizing codon replacement; Eliminate its NdeI of inner 10; Ecoli I; NotI; The restriction enzyme site of nucleic acid restriction endonuclease such as SacI and BamHI obtains the Zebra fish egg yolk protein origin 1 gene nvtg1 of new artificial reconstructed design, eliminates its inner NdeI; Ecoli I; NotI, SacI, restriction enzyme sites such as BamHI; Sequence is shown in SEQ ID NO.1; Total length 4095bp, the upper reaches contain Nde I restriction enzyme site CATATG, and downstream contain SalI restriction enzyme site GTCGAC.

Synthetic and the clone of embodiment 2 nvtg1 genes

Because the nvtg1 gene is compared with former vtg1 gene; Change bigger; Be not easy to use RT-PCR technology and site-directed mutagenesis technique; Therefore use chemical synthesis or the conventional synthetic nvtg1 gene of Over-Lap round pcr; Synthetic nvtg1 gene is connected with the PMD18T carrier; Transform JM109; The converted product coating contains the LB flat board of 50 μ g/mL penbritins; Through 37 ℃ of overnight incubation, obtain mono-clonal transformant nvtg1/PMD18T/JM109, identify through bacterium colony PCR; Plasmid enzyme restriction is identified and order-checking is identified; Show that new synthetic nvtg1 full length gene is 4095bp, 1364 amino acid of encoding, the sequence and the implementation sequence of mensuration are identical.

Embodiment 3 expresses the proteic vector construction of VTG

The nvtg1 gene is preferably pET-28a (+) at the carrier of expression in escherichia coli; This carrier has the His-tag label; PET-28a (+) plasmid and nvtg1/PMD18T are cut with Nde I and Sac I; Enzyme is cut product and is cut the glue recovery; Connect with the T4 ligase enzyme again; Connect product Transformed E .coli JM109 competent cell; Through 37 ℃ of overnight incubation; Select transformant; Identify through bacterium colony PCR; Plasmid enzyme restriction is identified and order-checking is identified, preserves nvtg1/pET-28a (+)/E.coli JM109 bacterium of identifying, it is subsequent use to extract nvtg1/pET-28a (+).

Embodiment 4 expresses the proteic engineering bacteria of VTG and makes up

With plasmid nvtg1/pET-28a (+) transformed into escherichia coli host BL21 (DE3), dull and stereotyped at the LB of 100 μ g/mL penbritins again, through 37 ℃ of overnight incubation, identify through bacterium colony PCR, obtain mono-clonal transformant nvtg1/pET-28a (+)/BL21 (DE3).

Proteic fermentative production of embodiment 5 VTG and separation and purification

1, fermentative production

Nvtg1/pET-28a (+)/BL21 (DE3) is seeded in the LB substratum 37 ℃ of liquid culture spends the night, TB (glycerine 5g/L, peptone 12g/L, yeast extract paste 24g/L, K are inserted in the back ₂HPO ₄12.54g/L, KH ₂PO ₄2.31g/L) fermentation broth, cultivate bacterium OD for 37 ℃ ₆₀₀To 0.8, to induce with 0.5mM IPTG (isopropylthio β D galactoside), 25-37 ℃ of cultivation produced enzyme and reached about 3mg/mL during 24h.

2, separation and purification

A certain amount of medium centrifugal with after expressing obtains cell, with the 10mM Tris-HCL of 1/10 nutrient solution volume; Ph7.5; Clean 3 times, then under condition of ice bath, at the 10mM Tris-HCL of 1/10 nutrient solution volume; Ph7.5; In, with the ripple fragmentation of overfulfiling a production target, working conditions is 10S work; 20S stagnates, and 30min overfulfils a production target.10000rpm, 4 ℃, centrifugal 20min obtains deposition.Deposition is cleaned 3 times with 1% Triton100, and then uses 10mM Tris-HCL, and Ph7.5 cleans; After the cleaning, 10000rpm, 4 ℃, centrifugal 20min obtains rVTG albumen.

Embodiment 6 SDS PAGE and Western identify recombinant expressed VTG (rVTG)

SDS PAGE program with conventional prepares 2 protein electrophoresis glue, behind the electrophoresis, does another piece of coomassie brilliant blue staining for one and is western blotting.Deposition condition: 80V 40min; 150V 50min.After electrophoresis finished, glue coomassie brilliant blue staining 2-4h, another piece were wester blotting, changeed the film condition: 100V, 90min; Film changes into to soak after the merit and seals 1h among the 5%BSA, anti-(1: 1000) liquid reaction 60min, TTBS washing 6 times, each 10min, reaction (1: 50000) 60min in two anti-(1: 250000) and HRP-linking agent reagent, TTBS washing 6 times, each 10min; 1: 1 mixing of chemoluminescence cross-linking reagent keeps in Dark Place, and 5min takes pictures.Western is positive, identifies that VTG is by recombinant expressed and have an antigenicity.

The application of embodiment 7 nvtg1 genes in environment measuring and food inspection and medical research and medical diagnosis on disease

Express engineering bacteria with the gene constructed VTG of nvtg1, through fermentative production VTG albumen.VTG albumen is that the important biomolecule of environment measuring and food inspection and medical research and medical diagnosis on disease is learned reagent.Utilize rVTG to replace natural VTG (nVTG) albumen as the mark article, based on the Elisa method, but the biological intravital VTG protein content of detection by quantitative, for organism internal hormone status evaluation provides basis.

Be understandable that, concerning those of ordinary skills, can be equal to replacement or change according to technical scheme of the present invention and inventive concept thereof, and all these changes or replacement all should belong to the protection domain of the appended claim of the present invention.

Claims (9)

1. the zebra fish of You Huaing (Danio rerio) vitellogenin genes nvtg1 is characterized in that nucleotide sequence is shown in SEQ ID NO.1.

2. gene nvtg1 according to claim 1 is characterized in that said nucleotide sequence uses colibacillary preferences codon.

3. the expression vector that contains the new gene nvtg1 of the said vitellogenin of claim 1.

4. expression vector according to claim 3 is characterized in that, said expression vector is an expression vector in the born of the same parents.

5. expression vector according to claim 4 is characterized in that being skeleton with pET-28a (+) preferably.

6. contain the new gene nvtg1 of the said vitellogenin of claim 1 or the transgenic cell line of 5 described expression vectors.

7. contain the new gene nvtg1 of the said vitellogenin of claim 1 or the genetic engineering bacterium of 5 described expression vectors.

8. genetic engineering bacterium according to claim 7 is characterized in that being preferably e. coli bl21 (DE3).

9. the new gene nvtg1 of the said vitellogenin of claim 1 or the 5 described expression vectors application in environmental inspection, food inspection, medical research and vitellogenin antibody preparation are produced.

Images