CN105543254A - Codon optimized beta-galactosidase gene and application thereof - Google Patents
Codon optimized beta-galactosidase gene and application thereof Download PDFInfo
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- CN105543254A CN105543254A CN201610056474.7A CN201610056474A CN105543254A CN 105543254 A CN105543254 A CN 105543254A CN 201610056474 A CN201610056474 A CN 201610056474A CN 105543254 A CN105543254 A CN 105543254A
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- 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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- C12Y302/01—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 codon optimized beta-galactosidase gene and an application thereof and belongs to the technical field of biochemistry and molecular biology. The nucleotide sequence of beta-galactosidase is shown as a sequence table SEQ ID NO.1. According to the preference of relevant high-expression protein codons in cow milk, codons of the bacterium-derived beta-galactosidase gene are optimized. The optimized OLacZ gene is verified in mouse mammary epithelial cells, and it is proved that the expression quantity of the gene is remarkably increased by about 3.7 times compared with that of a wild LacZ gene. The codon optimized OLacZ gene can be better effectively expressed in mammary tissue of a mother mouse in a lactation period. Beta-galactosidase serves as a reporter gene and is efficiently expressed in cow mammary tissue, and the effective tool gene is provided for further developing a mammary gland bioreactor.
Description
Technical field
The invention belongs to Biochemistry and Molecular Biology technical field, be specifically related to a kind of codon optimized beta-galactosidase gene and application thereof.
Background technology
LacZ gene is a gene in E coli lac operon, the tetramer that beta-galactosidase gene is made up of four subunits, can the hydrolysis of catalysing lactose.Beta-galactosidase enzymes is more stable, with X-Gal be substrate dye time, in blue, be convenient to detect and observe.LacZ gene is the reporter gene of widespread use in molecular biology and genetically engineered, detects the expressed in situ of albumen especially for histochemical stain, and the basic nontoxicity of the survival and growth of its expression product to cell is done.But the detection sensitivity of LacZ gene is not high, its detected result is subject to the impact of the many factors such as genetic expression, dyeing.
Genetic code has 64 kinds, but most biological tendency is in utilizing the part in these codons.Those are called best codon (optimalcodons) by what the most frequently utilize, utilize preference codon and avoid utilization ratio low or rare codon synthetic gene, are called as codon optimized.Due to the beta-galactosidase gene (LacZ) of bacterial origin in eukaryotic cell transcribe and translate insufficient, the preference that contriver uses according to the codon of those high expression level albumen in milk, attempts carrying out corresponding optimization to the codon of coding LacZ gene.To reaching at mammary gland specific high-efficiency expression through codon optimized LacZ gene (OlacZ), it not only can be able to high expression effectively in mammalian cell, and has mammary gland-specific in certain degree.That is at mammary epithelial cell, this can not in the cell of effective expression LacZ, can be able to express more fully originally.Yet there are no can in bovine mammary gland the codon optimized expressional scheme of the LacZ gene of high expression.
Summary of the invention
The present invention is directed to the defect of prior art, propose a kind of codon optimized beta-galactosidase gene and the application in galactophore biological reactor thereof.
A codon optimized beta-galactosidase gene, the nucleotide sequence of this beta-galactosidase gene is as shown in sequence table SEQ IDNO.1.
Containing the recombinant vectors of above-mentioned beta-galactosidase gene.
Containing the application in the transgenic engineering of above-mentioned recombinant vectors.
The application of above-mentioned codon optimized beta-galactosidase gene in galactophore biological reactor.
Beneficial effect of the present invention: the codon of the present invention to the beta-galactosidase gene (LacZ) of bacterial origin is optimized.The present invention demonstrates codon optimized effect, the OLacZ gene of the LacZ gene do not optimized and optimization is respectively charged into (promotor is pCMV) in pcDNA3.1 carrier for expression of eukaryon, and transfected mammary epithelial cell (HC11), the expression effect after protein level detects beta-galactosidase gene optimization is 3.7 times of former LacZ gene.On this basis, transgenic mice is prepared respectively with above-mentioned carrier.In female mouse lactation period, detect the beta-galactosidase enzymes expression activity of each internal organs, and then find that OLacZ has special optimizations of certain mammary gland and is inclined to.
Accompanying drawing explanation
Fig. 1 is the HC11 cell beta-galactosidase enzymes native staining of transfection different carriers.
Fig. 2 is the expression optimizing OLacZ and wild-type LacZ gene, mark " * * ": P<0.01, has significant difference.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention will be further described.
In following embodiment, plasmid pcDNA3.1-LacZ (pCMV-LacZ), pcDNA3.1-OLacZ (pCMV-OLacZ) are preserved by contriver.Mammary gland of mouse epithelial cell (HC11) purchased from American ATCC (AmericanTypeCultureCollection) cell bank.Synthesized by Invitrogen company through codon optimized OLacZ gene.
0.25% trypsinase, human epidermal growth factor, DMEM-F12 substratum, foetal calf serum are purchased from GibcoBRL company; Insulin human available from Sigma; Beta-galactosidase enzymes native staining test kit is purchased from green skies biotechnology research institute; LipofectamineTM2000 transfection reagent is purchased from Invitrogen company.
The optimization design of embodiment 1 beta-galactosidase gene codon and synthesis
The albumen that in milk, content is the highest is respectively α s1-casein (caseinalphas1, CSN1S1), α s2-casein (caseinalphas2, CSN1S2), beta-casein (caseinbeta, CSN2), K-casein (caseinkappa, CSN3), beta-lactoglobulin (beta-lactoglobulin), alpha-lactalbumin (lactalbumin, alpha-LALBA), cow's milk serum albumin (albumin, ALB) etc.The content of these albumen in milk is as table 1.Weight shared by several albumen that content in milk is the highest, and the codon usage frequency of the gene of these albumen of encoding in bovine mammary gland and frequency calculate, select best codon (see table 2), LacZ gene 1025 amino acid whose codons are replaced one by one.Gene after optimizing is called OLacZ (optimizedLacZ).Blast comparison codon optimized front and back nucleotide sequence, similarity is 77%, and aminoacid sequence before and after optimizing, similarity is 100%.
7 kinds of protein that in table 1 milk, content is the highest
Table 2 bovine mammary gland optimal codon table
Embodiment 2pCMV-LacZ and pCMV-OLacZ plasmid be transfection HC11 cell respectively
HC11 cell is seeded to 24 orifice plates, treats that cell grows to dull and stereotyped 70% density.Use LipofectamineTM2000, by liposome: plasmid pCMV-LacZ or pCMV-OLacZ is carried out the unsaturated transfection of same ratio to HC11 cell by DNA=2.5:1 (μ L/ μ g) respectively.At 37 DEG C, 5%CO
2change complete culture solution after cultivating 6h under condition to continue to cultivate.
After plasmid pCMV-LacZ, pCMV-OLacZ transfectional cell 48h, carry out cell dyeing according to beta-galactosidase enzymes native staining test kit specification sheets.Plasmid transfection enters cell, cell expressing beta-galactosidase gene (OLacZ).Take X-Gal as substrate, mazarine product can be generated under beta-galactosidase enzymes catalysis, thus be easy under an optical microscope observe become blue cell.Hatch 2h or longer time, observe counting at ordinary optical microscope for 37 DEG C.
Cell in-situ dyeing is carried out by after pCMV-LacZ, pCMV-OLacZ carrier transfection HC11 cell, observe under an optical microscope and take pictures (Fig. 1), statistics blue cell quantity, uses SPSS16.0 independently to repeat experiment to three times and carries out one-way analysis of variance.Result display (table 3, Fig. 2): in HC11 cell, the expression level of the LacZ gene (OLacZ) through optimizing is 3.7 times without the LacZ gene optimized, and P<0.05 (table 4), has significant difference.
Table 3 beta-galactosidase enzymes native staining method blue cell counts
Table 4 optimizes OLacZ and wild-type LacZ test results one-way analysis of variance
Embodiment 3 shows and in certain degree, embodies the trend with mammary gland-specific through codon optimized OLacZ in mouse level
By pCMV-LacZ and pCMV-OLacZ carrier DNA respectively through mouse fertilized egg procaryotic injection, transplanting and obtain several family turning pCMV-LacZ and pCMV-OLacZ DNA murine.Through mating, pregnancy, farrowing, treat about 10 days lactation period, put to death female mouse, after perfusion, get each internal organs.The betagalactosidase activity (green skies company) of each transgenic mice unit weight internal organs is detected by ONPG standard measure.Detected result display has the special optimization trend of certain mammary gland (female mouse=4 of pCMV-LacZ, female mouse=6 of pCMV-OLacZ) (see table 5) through the codon optimized OLacZ DNA murine that turns.
The each internal organs unit organization of table 5 lactational pCMV-LacZ and pCMV-OLacZ transgenic mice expresses the situation of beta-galactosidase enzymes
According to the preferences of high expression level albumen codon relevant in cow's milk juice, the codon of contriver to the beta-galactosidase gene (LacZ) of bacterial origin is optimized.OLacZ expressing gene after optimization is verified in mammary gland of mouse epithelial cell (HC11), proves that it significantly improves relative to the LacZ gene expression amount of wild-type, about 3.7 times.On this basis, contriver, with above-mentioned two kinds of genophores, prepares transgenic mice respectively.By detecting and compare the betagalactosidase activity of each internal organs of female mouse lactation period, determine, through codon optimized OLacZ gene, whether there is the special tendency of mammary gland.Result shows through codon optimized OLacZ gene in the mammary tissue of female mouse lactation period, more can effective expression.Beta-galactosidase enzymes is as a kind of reporter gene, and the high expression in bovine mammary gland tissue, for developing galactophore biological reactor further, provides a kind of effective instrument gene.
Claims (4)
1. a codon optimized beta-galactosidase gene, is characterized in that, the nucleotide sequence of this beta-galactosidase gene is as shown in sequence table SEQ IDNO.1.
2. containing the recombinant vectors of beta-galactosidase gene described in claim 1.
3. containing the transgenic engineering product of recombinant vectors described in claim 2.
4. the application of beta-galactosidase gene codon optimized described in claim 1 in galactophore biological reactor.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1269133A (en) * | 2000-04-25 | 2000-10-11 | 安靓 | Establishment of transgenosis animal or mammal reactor by means of intracorporeal embryo dry-cell transfection process |
| CN103898157A (en) * | 2012-12-24 | 2014-07-02 | 上海市儿童医院 | Method for producing human serum albumin by using animal mammary gland and expression vector thereof |
| CN103911393A (en) * | 2012-12-31 | 2014-07-09 | 上海滔滔转基因工程股份有限公司 | Construction and application of mammary specific expression vector for human transferrin |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1269133A (en) * | 2000-04-25 | 2000-10-11 | 安靓 | Establishment of transgenosis animal or mammal reactor by means of intracorporeal embryo dry-cell transfection process |
| CN103898157A (en) * | 2012-12-24 | 2014-07-02 | 上海市儿童医院 | Method for producing human serum albumin by using animal mammary gland and expression vector thereof |
| CN103911393A (en) * | 2012-12-31 | 2014-07-09 | 上海滔滔转基因工程股份有限公司 | Construction and application of mammary specific expression vector for human transferrin |
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| POEN, S.L., ET AL.: "Genbank accession number:EF136884.1", 《GENBANK》 * |
| 冷春玲: "酸性β－半乳糖苷酶基因密码子优化及高效表达", 《辽东学院学报( 自然科学版)》 * |
| 张斯敏 等: "乳腺生物反应器特异高效表达载体的构建", 《中国生物工程杂志》 * |
| 张莉 等: "牛αS1-酪蛋白5’调控序列驱动人α-LA 基因与LacZ 基因在牛乳腺上皮细胞中的表达", 《生物化学与生物物理进展》 * |
| 张骞 等: "乳腺上皮细胞的研究及应用", 《中国牧业通讯》 * |
| 杜威: "嗜热菌β-糖苷酶基因乳腺特异性表达载体的构建、转染及鉴定", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
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