CN102876693A - Xylanase gene xynB as well as expression vector and application of Xylanase gene xynB - Google Patents
Xylanase gene xynB as well as expression vector and application of Xylanase gene xynB Download PDFInfo
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Abstract
The invention relates to the technical field of biology, in particular to a xylanase gene xynB as well as an expression vector and an application of the xylanase gene xynB, and aims at solving the problem of low xynB gene expression quantity in the prior art. The xylanase gene xynB provided by the invention has the nucleotide sequence shown as SEQ ID NO:1 or SEQ ID NO:2. The xylanase expression vector contains the xylanase gene xynB. According to a method for improving the xynB gene expression quantity, the xylanase expression vector is transferred into HEK293A cells. The expression vector containing rare codons is used for the transfection of the HEK293A cells, so the expression quantity of the xynB gene is obviously improved, and an effective solution is provided for improving the expression quantity of the exogenous gene in transgenic animals.
Description
Technical field
The present invention relates to biological technical field, be specifically related to a kind of xylanase gene xynB and expression vector and application.
Background technology
The low situation about even not expressing of gene expression amount often appears during the heterogenous expression goal gene, cause this result's reason to have multiple, it is different to each Codon usage frequency in the synonym that one of them very important reason is exactly different plant species or different genes, and namely the preferences to codon is different.In the gene translation process, the codon that frequency of utilization is the highest is called as best codon (Optimal codons), and those frequencies of utilization are lower in addition under normal circumstances obsolete codon be called as rare codon (Rare codons), this phenomenon is codon preference.In order to eliminate the codon preference to the impact of exogenous gene expression, researcher has been attempted respectively multiple optimization method.
Can be divided into for the optimization of host cell with for the optimization of goal gene according to the object difference of optimizing.It is rare tRNA that the optimization of host cell is mainly referred to improve artificially the tRNA(that rare codon is corresponding in the host cell) abundance, namely in host cell, import the gene of expressing rare tRNA artificially, improve the abundance of specific rare tRNA in the host cell with the mode of crossing expression.Verified this mode can improve protein expression amount (Griswold really, Mahmood et al. 2003), but also there are many defectives in this method, can not get correct modification (Kurland and Gallant 1996 such as the tRNA of the metabolism pressure that increases the weight of host cell, heterogenous expression; Gustafsson, Govindarajan et al. 2004) and waste time and energy etc.And the optimal way of another kind is carried out for goal gene, has multiple different mode to implement to optimize in this method, but is the expression amount that will improve by the best codon of introducing host species in the different loci of gene gene on the whole.This one of them comparatively the method for " extremely " be the method that is known as " the corresponding codon of a seed amino acid ", this method is under the prerequisite that does not change protein amino acid sequence, all codons in the foreign gene encoding sequence all are optimized for the best codon of host species, studies show that these class methods really expression amount of foreign gene is improved (Zolotukhin, Potter et al. 1996; Marlatt, Spratt et al. 2010).But simultaneously also the someone proposes viewpoint and thinks that this change of former sequence being carried out on a large scale might change the mechanism (Griswold, Mahmood et al. 2003) of gene expression regulation.It should be noted that in addition, although each species can be selected the best codon of oneself in the process of evolving, but each species is not abandoned rare codon yet simultaneously, someone thinks in the process of evolving, the pressure of selecting can impel biospecific ground to select some rare codons, Singh etc. have confirmed this theory (Singh, Bauer DuMont et al. 2007) when the research fruit bat.The result that this selection is evolved has good enlightenment effect when optimizing the gene of heterogenous expression, not hard to imagine, if adopt the optimization method of " the corresponding codon of a seed amino acid " that gene is optimized, one can bring very large metabolism pressure to host cell, the fast synthetic rising that may cause the resultant fault rate of two this foreign proteins, perhaps the modification course of processing of polypeptide after synthetic is affected, such as can not normally folding etc.Introduce rare codon in the gene fragment of the suppositions such as Hershberg aminoacid sequence between coding synthetic proteins secondary structure territory, the resultant velocity of protein is slowed down a little at the rare codon place, impel synthetic polypeptide more fully folding (Hershberg and Petrov 2008), also can reduce simultaneously the metabolism pressure of host cell, the false folding of avoiding protein to produce because resultant velocity is too fast.
But in actually operating, for different concrete genes, also have a lot of uncertain places, same method is optimized different genes, the result who obtains is completely different, after such as Marlatt etc. the gene order of people's S100 albumen being optimized in this way, at expression in escherichia coli, its expression amount is than (the Marlatt that is significantly increased before optimizing, Spratt et al. 2010), can not increase the expression amount (Griswold, Mahmood et al. 2003) of causing root rot disease of Medicago sativa bacterium Cutinase albumen in E.coli but this optimal way is found in the experiment of Griswold etc., its precise mechanism there is no final conclusion.Particularly to the expression of xynB gene, not yet there are effective means or concrete expression vector to realize, the expression amount of xynB gene can not be improved, and tracing it to its cause is present people and does not know that codon is the level which type of mechanism to affect genetic expression by.
Summary of the invention
The objective of the invention is to overcome the low defective of xynB gene expression amount in the prior art, a kind of xylanase gene is provided
XynBAnd expression vector, to improve the expression amount of xynB gene.
For achieving the above object, the invention provides a kind of xylanase gene
XynB, its nucleotide sequence is shown in SEQ ID NO:1 or SEQ ID NO:2.
The present invention also provides a kind of zytase expression vector, and it contains above-mentioned xylanase gene
XynB, be specially zytase expression vector xynB-G249A or xynB-G489A.
Based on above-mentioned expression vector, the present invention also provides a kind of method of expression amount of the xynB of raising gene, specifically above-mentioned zytase expression vector is changed in the HEK293A cell (mice embryonic nephrocyte).
The preparation method of above-mentioned zytase expression vector is as follows.At first, mode according to " the corresponding codon of a seed amino acid ", codon-bias according to the mankind synthesizes again to zytase xynB gene, obtained zytase expression vector xynB-11Opt, wherein the codon in the xynB gene coded sequence all is human best codon, and then rare codon is introduced by the mode of point mutation in two sites in the selected xynB gene.Wherein selected mutational site lays respectively at the 249th bit base and the 489th bit base of xynB gene, obtain respectively zytase expression vector xynB-G249A and xynB-G489A, the former link zone of amino acid between two β-pleated sheet structures of zytase secondary structure corresponding to rare codon of containing wherein, and the link zone of amino acid between β-pleated sheet structure and α spiral corresponding to the rare codon that the latter is contained.Selected two codons corresponding to mutational site are the synthetic leucine of CTG(coding in carrier xynB-11Opt), point mutation by G → A makes CTG be substituted by CTA, concerning the mankind, the frequency of utilization of CTG is 39.6 ‰, and the frequency of utilization of CTA is 7.2 ‰.
Compared with prior art, the present invention has following beneficial effect:
The present invention introduces rare codon at the specific site of the xynB gene of optimizing through " the corresponding codon of a seed amino acid " mode, use respectively the expression vector transfection HEK293A cell that contains rare codon and do not contain rare codon, discovery has been introduced after the rare codon, the expression amount of xynB gene is significantly improved, wherein 249 rare codon makes gene expression amount improve about 30%, and 489 rare codon makes the about 60%(of gene expression amount raising know by the fluorescence intensity among the comparison Flow cytometry result), provide effective solution for improving the expression amount of foreign gene in transgenic animal.
Description of drawings
Fig. 1 zytase xynB-G249A(schemes A) and xynB-G489A(figure B) space structure figure; Black arrow indication mutational site, C and N represent respectively C-terminal and the N-terminal of albumen; The different different secondary structures of shape representative, wherein band shape represents β-pleated sheet structure, and dark spiral represents the α spiral, dark column representative curling (Coil), light column represents corner (Turn).
Fig. 2 is the as a result figure of flow cytometer detection behind the transient transfection cell, ordinate zou represents fluorescence intensity among the figure, X-coordinate xynb-11Opt, xynb-G249A, xynb-G489A represent to use respectively corresponding plasmid transfection cell, and three alphabetical a, b, c represent that utmost point significant difference is arranged between the three (P＜0.01) among the figure.
Fig. 3 is the Western blot detected result figure of total protein of cell after the extraction transfection; Its at the middle and upper levels band representative be the albumen of gene xynB and GFP amalgamation and expression, lower floor is the protein band of house-keeping gene GAPDH, xynb-11Opt, xynb-G249A, xynb-G489A represent respectively the total protein with corresponding plasmid transfection cell.
Fig. 4 is the as a result figure of quantitative PCR detection genetic expression; Wherein, xynb-11Opt, xynb-G249A, xynb-G489A represent respectively with corresponding plasmid transfection cell, P〉0.05.
Embodiment
The employed carrier of the following embodiment of the present invention, bacterial classification, reagent and source thereof:
The pEGFP-N1 carrier is available from Clontech company; The Host Strains bacillus coli DH 5 alpha is available from TAKARA company; PBluescript II SK+ carrier is available from Stratagene company; HEK293A clone is available from Invitrogen company; Synthetic and the sequencing of sequence and primer is finished by SangonBiotech company; Taq enzyme, T4DNA ligase enzyme, restriction endonuclease are all from Dalian TaKaRa company; Western blot detects all antibody of usefulness all available from Abcam company, contains the ECL luminescent solution of HRP substrate available from Thermo company; The point mutation test kit is available from Stratagene company; Cell culture medium DMEM and foetal calf serum are available from Gibico company; The reverse transcription test kit is available from Promega company; Liposome Lipofectamine 2000 and RNA extract reagent TRIzol all available from Invitrogen company; The protein extraction test kit is available from Kai Ji company.The normal experiment operation stepss such as enzyme is cut, connected, recovery, conversion, pcr amplification see " molecular cloning " for details.
Embodiment 1The selection in mutational site
(Schwede 2003 to utilize SWISS-MODEL website (http://swissmodel.expasy.org/); Arnold, Bordoli et al. 2006) space structure of on-line prediction zytase, template is albumin X YNL(PDB-ID:1hix), learn that the secondary structure in each monomer has 1 α spiral, 14 β-pleated sheet structures and 14 loop zones, therefore two mutational sites are designed respectively between near the α spiral of C-terminal and β-pleated sheet structure and two β-pleated sheet structures between (such as Fig. 1), i.e. the 249th bit base of xynB gene and the 489th bit base.
Embodiment 2The structure of zytase expression vector
1.1 do not contain the zytase expression vector establishment of rare codon
Download zytase protein sequence (obtain number be ABF02144) from GenBank, counter its nucleotide sequence of releasing of method according to " the corresponding codon of a seed amino acid ", whole codons in the encoding sequence are human best codon, the signal peptide of itself is replaced by the β of ox-casein signal peptide, terminator codon also is removed, the anti-sequence (shown in SEQ ID NO:3) of releasing is sent synthetic by SangonBiotech company (Shanghai, China) and is cloned in the pBluescript II SK+ carrier.Cut with Nhe I and two restriction enzyme site enzymes of BamH I, the fragment subclone that reclaims is to the pEGFP-NI carrier, obtain zytase expression vector xynB-11Opt, wherein contain and derive from Streptomyces olivaceoviridis A1(Streptomyces olivaceoviridis A1) xylanase gene, the nucleotide sequence of xynB gene is shown in SEQ ID NO:3 in this xynB-11Opt plasmid.
1.2 contain the zytase expression vector establishment of rare codon
The introducing of rare codon is finished by simple point mutation, principle is: for selected site design mutant primer, nucleotide site after the sudden change is included in the primer sequence, take the xynB-11OPt plasmid as template, carry out PCR with the Pfu archaeal dna polymerase, then the PCR product being carried out enzyme with the DpnI enzyme cuts, this enzyme methylated DNA chain of can degrading specifically, modify and degraded specifically by the DpnI enzyme because contained to methylate by the synthetic template sequence of E.coli DH5 α, synthetic gene order in the PCR process because do not contain methylates and is retained, and only has the plasmid after the sudden change in the final product.
Concrete steps are as follows: 1) carry out PCR take the xynB-11Op plasmid as template, the xynB893F in the table 1 and xynB893R are as primer.According to each component in the table 2 adding system, then carry out PCR by the program in the table 3.After the PCR EP (end of program), add 1 μ l enzyme DpnI in the reaction system, behind the mixing, place 1h for 37 ℃, the PCR product that obtain this moment can 4 ℃ of preservations; 2) with 1) in products therefrom be transformed in the E.coli DH5 α competent cell according to conventional heat shock mode, by Amp resistance screening positive colony, method by gel electrophoresis behind the bacterium liquid PCR detects whether the appearance of purpose band is arranged, if be positive colony, otherwise be false positive, should reject.1-2 positive colony sample presentation order-checking of each sample picking.With the clone's enlarged culturing that checks order correct, spend the intracellular toxin plasmid extraction kit and extract plasmid, measure plasmid concentration, and after detecting quality with agarose gel electrophoresis, qualified sample-20 ℃ saves backup, and namely obtains the xynB-G249A carrier.
XynB1133F and xynB1133R are as primer in the table 1, and operation can obtain the xynB-G489A carrier according to the method described above.
The tabulation of table 1 primer
Table 2 simple point mutation PCR reaction system
Table 3 simple point mutation PCR response procedures
Embodiment 3HEK293A cell transfecting and genetic expression detect
2.1 cell cultures and transient transfection
The HEK293A cell is seeded in the 6 porocyte culture plates, when in containing the DMEM substratum of 10% foetal calf serum, being cultured to 70-80% degree of converging, to specifications requirement changes zytase expression vector xynB-11OPt, xynB-G249A and xynB-G489A respectively in the HEK293A cell with Lipofectamine 2000 liposomes.
2.2 flow cytometry and Western blot gene expression detection
Get the HEK293A cell behind the transfection 48h, trysinization is collected in the 1.5ml centrifuge tube.The average green fluorescence intensity of 10000 cells of flow cytometer detection.Flow cytometry result shows and introduces that the gene expression amount on the translation skill significantly improves after the rare codon, wherein the XYNB gene expression amount improves 30% approximately in the xynB-G249A carrier, and the XYNB gene expression amount improves 60%(Fig. 2 approximately in the xynB-G489A carrier).
The HEK293A cell of transfection is cultivated 48h in incubator after, the total protein that extracts cell with the protein extraction test kit of Kai Ji company is used for doing Western blot hybridization.The primary antibodie of hybridization usefulness is the anti-GFP polyclonal antibody of rabbit, and two anti-are the mouse-anti rabbit antibody of horseradish peroxidase, and house-keeping gene is selected glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH).Fig. 3 discal patch band is carried out gray scale scanning to be learnt, the xynB-11OPt respective value is 189.25, the xynB-G249A respective value is that the ratio of 255.15(and xynB-11Opt respective value is 1.35), the xynB-G489A respective value is that the ratio of 273.41(and xynB-G489A respective value is 1.44), the result who as seen obtains and the result of flow cytometry substantially coincide (Fig. 3).
2.3 quantitative PCR detection genetic expression
Cultivate the HEK293A cell of 48h after the transfection, extract cell total rna with Trizol, reverse transcription obtains detecting with the method for quantitative PCR behind the cDNA expression of gene, and house-keeping gene selects the GAPDH(the primer to see Table 1).Detected result shows, rare codon arranged to the expression of gene do not make significant difference (Fig. 4) at transcriptional level.
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Claims (3)
1. an xylanase gene xynB is characterized in that its nucleotide sequence is shown in SEQ ID NO:1 or SEQ ID NO:2.
2. a zytase expression vector is characterized in that containing the described xylanase gene xynB of claim 1.
3. a method that improves the xynB gene expression amount is characterized in that the described zytase expression vector of claim 2 is changed in the HEK293A cell.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10131895B2 (en) | 2013-03-12 | 2018-11-20 | Basf Enzymes Llc | Genes with codon mutations encoding xylanase |
Citations (3)
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|---|---|---|---|---|
| CN1451753A (en) * | 2003-05-21 | 2003-10-29 | 江南大学 | Technical scheme for increasing maricolous thermobacillus xylanase B gene expression level by large margin |
| WO2009045627A2 (en) * | 2007-10-03 | 2009-04-09 | Verenium Corporation | Xylanases, nucleic acids encoding them and methods for making and using them |
| CN102424828A (en) * | 2011-11-15 | 2012-04-25 | 南京林业大学 | Gene capable of increasing expression level of aspergillus niger xylanase, recombinant plasmid and host cell thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1451753A (en) * | 2003-05-21 | 2003-10-29 | 江南大学 | Technical scheme for increasing maricolous thermobacillus xylanase B gene expression level by large margin |
| WO2009045627A2 (en) * | 2007-10-03 | 2009-04-09 | Verenium Corporation | Xylanases, nucleic acids encoding them and methods for making and using them |
| CN102424828A (en) * | 2011-11-15 | 2012-04-25 | 南京林业大学 | Gene capable of increasing expression level of aspergillus niger xylanase, recombinant plasmid and host cell thereof |
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| YANG,H.M.ET AL.,: "GenBank:ABF02144.1", 《GENBANK》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10131895B2 (en) | 2013-03-12 | 2018-11-20 | Basf Enzymes Llc | Genes with codon mutations encoding xylanase |
| US10619142B2 (en) | 2013-03-12 | 2020-04-14 | Basf Enzymes Llc | Genes with codon mutations encoding xylanase |
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