CN105543254B - A kind of beta-galactosidase gene of codon optimization and its application - Google Patents
A kind of beta-galactosidase gene of codon optimization and its application Download PDFInfo
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- CN105543254B CN105543254B CN201610056474.7A CN201610056474A CN105543254B CN 105543254 B CN105543254 B CN 105543254B CN 201610056474 A CN201610056474 A CN 201610056474A CN 105543254 B CN105543254 B CN 105543254B
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2468—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on beta-galactose-glycoside bonds, e.g. carrageenases (3.2.1.83; 3.2.1.157); beta-agarase (3.2.1.81)
- C12N9/2471—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
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- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01023—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
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Abstract
The invention discloses a kind of beta-galactosidase gene for the codon optimization for belonging to Biochemistry and Molecular Biology technical field and its applications.The nucleotide sequence of the beta galactosidase is as shown in sequence table SEQ ID NO.1.The present invention optimizes the codon of the beta-galactosidase gene of bacterial origin according to the preferences of high expression albumen codon related in cow's milk juice.OLacZ gene after optimization is verified in mammary gland of mouse epithelial cell, it was demonstrated that and it is significantly improved relative to the LacZ gene expression amount of wild type, and about 3.7 times.OLacZ gene through codon optimization, more can effective expression in the breast tissue of lactation period female rat.For beta galactosidase as a kind of reporter gene, the high efficient expression in cow's milk glandular tissue provides a kind of effective tool gene further to develop galactophore biological reactor.
Description
Technical field
The invention belongs to Biochemistry and Molecular Biology technical fields, and in particular to a kind of β-gala of codon optimization
Glycosidase genes and its application.
Background technique
LacZ gene is a gene in E coli lac operon, and beta-galactosidase gene is by four subunits
The tetramer of composition, can catalysing lactose hydrolysis.Beta galactosidase is more stable, when with X-Gal being that substrate is dyed, is in
Blue, convenient for detection and observation.LacZ gene is widely applied reporter gene in molecular biology and genetic engineering, especially
For the expression in situ of histochemical stain detection albumen, expression product is substantially non-toxic to the survival and growth of cell to be made.
However the detection sensitivity of LacZ gene is not high, testing result is influenced by many factors such as gene expression, dyeing.
Genetic code has 64 kinds, but most biological tendencies are in utilizing a part in these codons.Those quilts
It is most frequent utilize be known as best codon (optimal codons), using preference codon and avoid utilization rate low or dilute
Some codons synthesize gene, referred to as codon optimization.Since the beta-galactosidase gene (LacZ) of bacterial origin is true
Transcription and translation in nucleus is insufficient, the preference that inventor uses according to the codon of those height expression albumen in milk,
It attempts accordingly to optimize the codon of coding LacZ gene.To reach in mammary gland specific high-efficiency expression through codon optimization
LacZ gene (OlacZ), it is not only able to effectively be able to high efficient expression in mammalian cells, and in certain journey
There is mammary gland-specific on degree.That is galactophore epithelial cell this originally can not in the cell of effective expression LacZ,
It can more fully hereinafter be expressed.Yet there are no can in bovine mammary gland the LacZ gene of high efficient expression codon optimization table
Up to scheme.
Summary of the invention
The present invention in view of the drawbacks of the prior art, proposes a kind of beta-galactosidase gene of codon optimization and its in cream
Application in gland bioreactor.
A kind of beta-galactosidase gene of codon optimization, the nucleotide sequence of the beta-galactosidase gene such as sequence
Shown in table SEQ ID NO.1.
Recombinant vector containing above-mentioned beta-galactosidase gene.
Application in transgenic engineering containing above-mentioned recombinant vector.
Application of the beta-galactosidase gene of above-mentioned codon optimization in galactophore biological reactor.
Beneficial effects of the present invention: the present invention carries out the codon of the beta-galactosidase gene (LacZ) of bacterial origin
Optimization.The present invention demonstrates the effect of codon optimization, and the LacZ gene being not optimised and the OLacZ gene of optimization are respectively charged into
In pcDNA3.1 carrier for expression of eukaryon (promoter pCMV), and mammary gland of mouse epithelial cell (HC11) is transfected, in protein level
Expression effect after detecting beta-galactosidase gene optimization is 3.7 times of original LacZ gene.On this basis, with above-mentioned carrier
Prepare transgenosis mouse respectively.In female rat lactation period, the beta galactosidase expression activity of each internal organs is detected, and then find
There is OLacZ certain mammary gland specifically to optimize tendency.
Detailed description of the invention
Fig. 1 is the HC11 cell beta galactosidase native staining for transfecting different carriers.
Fig. 2 is the expression for optimizing OLacZ and wild type LacZ gene, is marked " * * ": P < 0.01 has statistics poor
It is different.
Specific embodiment
The present invention will be further described in the following with reference to the drawings and specific embodiments.
Plasmid pcDNA3.1-LacZ (pCMV-LacZ), pcDNA3.1-OLacZ (pCMV-OLacZ) in following embodiments
It is saved by inventor.Mammary gland of mouse epithelial cell (HC11) is purchased from U.S. ATCC (American Type Culture
Collection) cell bank.OLacZ gene through codon optimization is synthesized by Invitrogen company.
0.25% trypsase, hEGF, DMEM-F12 culture medium, fetal calf serum are public purchased from GibcoBRL
Department；Actrapid monotard is purchased from Sigma company；Beta galactosidase native staining kit is purchased from green skies biotechnology research institute；
2000 transfection reagent of LipofectamineTM is purchased from Invitrogen company.
The optimization design and synthesis of 1 beta-galactosidase gene codon of embodiment
The highest albumen of content is respectively α s1- casein (casein alpha s1, CSN1S1), α s2- junket egg in milk
White (casein alpha s2, CSN1S2), beta-casein (casein beta, CSN2), K- casein (casein kappa,
CSN3), beta lactoglobulin (beta-lactoglobulin), alpha lactalbumin (lactalbumin, alpha-LALBA), cow's milk
Seralbumin (albumin, ALB) etc..Content of these albumen in milk such as table 1.It is highest several according to content in milk
The codon usage frequency and frequency that the gene of these albumen is encoded in weight shared by kind albumen and bovine mammary gland are counted
It calculates, selects best codon (being shown in Table 2), the codon of 1025 amino acid of LacZ gene is replaced one by one.After optimizing
Gene be known as OLacZ (optimized LacZ).Blast compares nucleotide sequence before and after codon optimization, and similitude is
77%, optimization front and back amino acid sequence, similitude 100%.
The highest 7 kinds of protein of content in 1 milk of table
2 bovine mammary gland optimal codon table of table
Embodiment 2pCMV-LacZ and pCMV-OLacZ plasmid transfect HC11 cell respectively
It is long to 70% density of plate to cell by HC11 cell inoculation to 24 orifice plates.Use LipofectamineTM
2000, by liposome: plasmid pCMV-LacZ or pCMV-OLacZ are carried out same ratio insatiable hunger by DNA=2.5:1 (μ L/ μ g) respectively
With transfection to HC11 cell.In 37 DEG C, 5%CO2Under the conditions of cultivate 6h after replacement complete culture solution continue to cultivate.
After plasmid pCMV-LacZ, pCMV-OLacZ transfect cell 48h, according to beta galactosidase native staining kit
Specification carries out cell dyeing.Plasmid transfection enters cell, and cell expresses beta-galactosidase gene (OLacZ).It is with X-Gal
Substrate can generate navy blue product under beta galactosidase catalysis, become to be readily observed under an optical microscope
Blue cell.37 DEG C of incubation 2h or longer time are observed in ordinary optical microscope and count.
Cell in-situ dyeing is carried out after pCMV-LacZ, pCMV-OLacZ carrier are transfected HC11 cell, in optical microphotograph
It under the microscope and takes pictures (Fig. 1), counts blue cell quantity, single factor test is carried out to independent repetition experiment three times with SPSS16.0
Variance analysis.As the result is shown (table 3, Fig. 2): in HC11 cell, the expression of optimized LacZ gene (OLacZ) is not
3.7 times of optimized LacZ gene, P < 0.05 (table 4) have significant difference.
3 beta galactosidase native staining method blue cell of table counts
Table 4 optimizes OLacZ and wild type LacZ test results one-way analysis of variance
Embodiment 3 shows that the OLacZ through codon optimization is embodied in certain degree with cream in mouse level
The trend of gland specificity
PCMV-LacZ and pCMV-OLacZ carrier DNA is obtained several through mouse fertilized egg procaryotic injection, transplanting respectively
Turn the family of pCMV-LacZ and pCMV-OLacZ DNA murine.Through mating, pregnancy, farrowing, to lactation period 10 days or so, put to death
Female rat takes each internal organs after perfusion.The beta galactose glycosides of each transgenic mice Unit Weight internal organs is detected by ONPG standard measure
Enzymatic activity (green skies company).Testing result shows that the OLacZ DNA murine that turns through codon optimization has certain mammary gland special
Different optimization trend (pCMV-LacZ female rat=4, pCMV-OLacZ female rat=6) (being shown in Table 5).
Each internal organs unit organization of the pCMV-LacZ and pCMV-OLacZ transgenic mice of 5 lactation period of table expresses beta galactose
The case where glycosides enzyme
According to the preferences of high expression albumen codon related in cow's milk juice, beta galactose glycosides of the inventor to bacterial origin
The codon of enzyme gene (LacZ) optimizes.OLacZ expressing gene after optimization is in mammary gland of mouse epithelial cell (HC11)
It is verified, it was demonstrated that it is significantly improved relative to the LacZ gene expression amount of wild type, and about 3.7 times.On this basis, it sends out
The above two genophore of bright people, difference prepare transgenosis mouse.By the β-for detecting and comparing each internal organs of lactation period female rat
Galactosidase activity, to determine whether the OLacZ gene through codon optimization there is mammary gland to be specifically inclined to.As the result is shown through close
The OLacZ gene of numeral optimization, more can effective expression in the breast tissue of lactation period female rat.Beta galactosidase is as a kind of
Reporter gene, the high efficient expression in cow's milk glandular tissue provide a kind of effective work further to develop galactophore biological reactor
Has gene.
Claims (3)
1. a kind of beta-galactosidase gene of codon optimization, which is characterized in that the nucleotide of the beta-galactosidase gene
Sequence is as shown in sequence table SEQ ID NO.1；
The method of the beta-galactosidase gene codon optimization are as follows: according to shared by the highest several albumen of content in milk
The codon usage frequency and frequency that the gene of these albumen is encoded in weight and bovine mammary gland are calculated, and are selected best close
Numeral；
The highest several albumen of content are α s1- casein, α s2- casein, beta-casein, K- casein, β-in the milk
Lactoglobulin, alpha lactalbumin and cow's milk seralbumin.
2. the recombinant vector containing beta-galactosidase gene described in claim 1.
3. application of the beta-galactosidase gene of codon optimization described in claim 1 in galactophore biological reactor.
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