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WO2012109788A1 - Procédé pour augmenter l'efficacité d'expression de protéine et vecteur d'expression à cet effet - Google Patents

Procédé pour augmenter l'efficacité d'expression de protéine et vecteur d'expression à cet effet Download PDF

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Publication number
WO2012109788A1
WO2012109788A1 PCT/CN2011/071030 CN2011071030W WO2012109788A1 WO 2012109788 A1 WO2012109788 A1 WO 2012109788A1 CN 2011071030 W CN2011071030 W CN 2011071030W WO 2012109788 A1 WO2012109788 A1 WO 2012109788A1
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Prior art keywords
seq
amino acid
protein
expression vector
efficiency
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Chinese (zh)
Inventor
张庆利
黄倢
刘笋
刘庆慧
史成银
梁艳
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Yellow Sea Fisheries Research Institute Chinese Academy of Fishery Sciences
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Yellow Sea Fisheries Research Institute Chinese Academy of Fishery Sciences
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Priority to CN201180001670.XA priority Critical patent/CN102333870B/zh
Priority to PCT/CN2011/071030 priority patent/WO2012109788A1/fr
Publication of WO2012109788A1 publication Critical patent/WO2012109788A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression

Definitions

  • the invention belongs to the field of genetic engineering technology, and has a method for improving the efficiency of protein «3 ⁇ 4" and a «3 ⁇ 4 carrier.
  • Sib is affected by many factors in the host cell, such as the nature of the gene of interest, transcription efficiency, mRNA stability, translation efficiency, and protein stability.
  • the characteristics of the target gene sequence such as rare codons, terminators, and secondary structures, often affect the surface of the protein. If the target group contains more rare codons, it is often a recombinant protein.
  • the table 3 ⁇ 4* is lower (Sorensen et al., 1989, J. Mol. Biol. 207, 365-377); if the rare codon set appears in 3 ⁇ 43 ⁇ 4, the situation is more serious (Chen and Inouye, 1990, Nucl) Acids Res.
  • the tRNA junction in the host cell reflects the codon usage of the host species mRNA; the rarity or lack of a sufficient tRNA may cause the translation to stop, and the sufficiency may result in translation. Delayed, pre-mature translation termination, translation of tree horses and distribution (Ikemura 1985, Mol. Biol. Evol. 2, 13-34); although the presence of a small number of rare codons usually does not have a large effect on the synthesis of the protein of interest, but if When a gene contains a string or a plurality of rare codons, the paste of the source protein is very low, rnm, so that the target gene contains too many rare codons.
  • the role of the vector is neglected, because it not only determines the copy number of the heavy granule, but also the recording efficiency and stability of the mRNA, and the effect of the slanting The stability of the recombinant protein.
  • a « said, the construction of the «3 ⁇ 4 carrier should meet the following requirements: (1) high protein surface, (2) a wide range of host applications, (3) the expression of the product is easy to purify.
  • Recombinant protein expression Volume is a priority indicator. How to build a high-efficiency 3 ⁇ 43 ⁇ 4 ⁇ 4 body has a lot of fiber, the research mostly focuses on the redrawing of the promoter, multiple cloning sites, stop codon, fusion protein «, replicon, screening feiB / reporter gene, etc. It is not yet possible to use some of the citric acid multi-password codons to add the ⁇ sequence to construct a high-efficiency carrier.
  • the object of the present invention is a method and a vector for improving protein efficiency, and the method or the vector of the present invention can improve the efficiency of exogenous host in a host, and obtain high efficiency of the target gene to make up for the prior art. insufficient.
  • a certain number of said target coding horses are introduced after the start codon of the target gene or the start codon of the carrier sequence in the recombination of the sputum phase (4 or
  • the above codons are the bribes of acid expansion.
  • These nucleotides encoding multiple codons «acids, which are rich in tRNA content, can participate in protein synthesis corresponding to S acid, thus avoiding the lack of tRNA in the target gene sequence or vector sequence.
  • the resulting mRNA translation, weighing or stopping has the initial and extension efficiency of the high translation, and greatly improves the efficiency of the target gene or the protein picked up.
  • the 3 ⁇ 4 ⁇ 4 carrier starts with ATG and contains a nucleotide of the MM" segment of the gestation of the woman.
  • the number of women in the specific volume of the leg spoon is not less than three.
  • the leg spoon body is the male body 3 ⁇ 43 ⁇ 43 ⁇ 4 body.
  • a method for increasing the efficiency of a gene of interest protein 3, which inserts a nucleoside encoding a specific succinct segment after the initiation codon ATG of the gene of interest, the levy is:
  • the specific column of the family 3 ⁇ 4 is X1X2... Xn, wherein X f3 ⁇ 4* has 4 or more codons of S acid, n is greater than or equal to 3;
  • the method of the present invention for the use of gene protein efficiency can be used not only Any gene of interest is used to increase its efficiency for the construction of vectors with high efficiency characteristics.
  • the present invention has the following method for improving the efficiency of the gene protein 3 ⁇ 4 ⁇ 4 by the present invention, and can be used for the 3 ⁇ 4it arbitrary target gene to increase the efficiency of the target protein by 2 to 5 times;
  • Example 1 constructs a high-efficiency fiber vector by inserting a nuclear salary column of the Kangma III constant-pay column after the start codon: a). Inserting the coding GlyGlySer (SEQ ID N0: 1) after the vector pGFPuv start codon The checklist:
  • a X-inch primer for the 3 ⁇ 4 pGFPuv vector was designed.
  • the upstream bow was PI (pGFPuv-S): GACATGA-GTAAAGGAGMGMC, and the downstream primer P2 (pGFPuv-A): GCGTTAnTGTAGAGCTCAT.
  • PI pGFPuv-S
  • pGFPuv-A the downstream primer P2
  • pGFPuv216-F designed upstream primer P3 (pGFPuv216-F): CTCATOGATATGACCATGATTAOG
  • at the 5, end are added to protect the bases CTC.
  • primers p5 and p6 for inserting the nucleic acid sequence encoding GlyGlySer upstream primer P5 (high-efficiency-F) were designed: TCTATCGATATGGGGGGTTCTATGATTAOGCCMGCTTGCA, containing Cla I restriction site 3 ⁇ 4 ⁇ codon ATG, downstream bow
  • P3 (pGFPuv216-F) : CTCATOGATATGACCATGATTACG
  • P4 (pGFPuv216-R) : GAGATCGATAGCTGTTTCCTGTG was used as the bowel I
  • the pGFPuv plasmid was used to clone the pGFPuv, so that the pGFPuv plasmid was cleaved at position 216, and both ends were added.
  • the enzyme cleavage site Cla I.
  • the PCR reaction was carried out in a total volume of 50 ⁇ l, using 2 ⁇ l of the pGFPuv plasmid as a template.
  • the reaction bovine was circulated after denaturation at 95 °C for 2 min, then denatured at 95 °C for 20 s, and annealed at 42 °C for 20 s, 72 °.
  • the linear p3p4GFPuv fragment was recovered by the electrophoresis of the sugar lake.
  • the p3p4pGFPuv fragment carrying the Cla I restriction site was ligated with restriction endonucleases Cla I and Hind III, and the fragment of 3. 3Kb after restriction enzyme digestion was recovered;
  • the p5p6 double-stranded DNA fragment was digested with the endonuclease Cla I and Hind III, and the fragment of about 50 bp was recovered by electrophoresis.
  • Two anterior ligatures were ligated, and ligated to col ⁇ ⁇ 5 ⁇ sensitized cells, and then coated with LB plate of 3 ⁇ 4 benzylpenicillin. After culture for 16 h, the single-sided 3 ⁇ 4 ⁇ microscopic microscopic examination was performed to confirm the positive clone.
  • the high-efficiency " ⁇ FPuv plasmid was transformed into Escherichia coli DH5a, cultured at 37 °C for 16 h, 3 ⁇ 4 ⁇ vine, 37, 220 rmf cultured to 0D600 ⁇ tS 1. 0, and extracted with 0MAGE plasmid in small amount.
  • the extracted heavy ISJ3 ⁇ 4 granulating was transferred into the bacterium DH5a into the fluorescent protein (using the pGFPuv vector plasmid as a control), and the fluorescence state was observed under the microscope, and the pronucleus of the fluorescing protein was subjected to 3 ⁇ 4 ⁇ 4.
  • a single clock with pGFPuv and high-efficiency " ⁇ FPuv heavy ⁇ -particles was inoculated into the LB liquid medium of the mixed sheep of mycin, cultured at 37 °C for 8-10 h, and then 13 ⁇ 4 species were added to the LB liquid medium of the mixed sheep.
  • the light was induced by IPTG with lm mol/L, and after induction at 28 °C for 5 h, the cells were collected by centrifugation, and the supernatant and precipitate were collected separately for SDS_PAGE.
  • the fluorescent protein was induced into the fiber, and the high-efficiency "GFPuv"
  • the amount of vector in the vector group was significantly higher than that of the control group pGFPuv; after induction of 3 ⁇ 4 ⁇ 4 GFP samples, the incomplete denaturing of Siff-PAGE was performed using the FujiFilm (LAS 3000) system, and the blue light was excited in GFP mode, GFP.
  • the protein band can be seen in bright green, and the other proteins ⁇ # less, play a role similar to Western-blot.
  • SM GlyGlySer a nuclear code of SM GlyGlySer to the start codon of the vector pGFPuv, which encodes three poly-codon acids and serines, respectively, and SM GlyGlySer, can increase the related protein expression of the vector by 3 More than one.
  • the nuclear intoxication column GGGGGTTCT can also be replaced by the nucleus, column, and other nuclear l ⁇ columns of GlyGlySer of SEQ ID N0: 3 ⁇ 6.
  • the nuclear rafts of the primers P5 and p6 are separated, so that the nuclear ⁇ column of the book code GlyGlySer is SEQ ID N0: 3-6 respiratory
  • the primer for P5 was changed to 7: TCTATCGATATGGGAGGTAGCATGATTAOGCCAAGCTTGCA; the underlined is the 3 ⁇ 4S sequence of Xie ⁇ ; the primer for P6 was changed to 8 : TGCMGCTTGGOGTAATCATGCTACCTCCCATATOGATAGA; wherein the underlined is the 3 ⁇ 4S sequence of ⁇ .
  • Amplification was bow I pGFPuv plasmid p3 and p4 P 3p4GFPuv fragment obtained, then p7, p8 bow
  • p7p8 was annealed to double-stranded DNA fragments
  • restriction enzymes were used Cla I and Hind III double digestion fragments P 3p4GFPuv And P 7p8 double-stranded DNA fragment, the restriction product was recovered and ligated, and after ligation to co7i DH5 a competent cells, a single colony was amplified by pl and p2 primers, positive clones were determined by electrophoresis, and positive clones were sequenced to ensure 3 ⁇ 43 ⁇ 43 ⁇ 4 ⁇
  • the column GGGGGTTCT has been inserted correctly after the start codon.
  • the heavy particle was named p7 P 8 efficient " ⁇ 1 ⁇ 2 ⁇ plasmid.
  • the P 7p8 high-efficiency " ⁇ FPuv plasmid was transferred into Bacillus DH5 a and cultured at 37 ° C for 16 h. After the culture was carried out, the single vine was expanded and the P7p8 efficient GFPuv plasmid was extracted. The extracted plasmid was transferred into Escherichia coli. DH5 a was inserted into the fluorescent protein *3 ⁇ 4, and the pGFPuv vector plasmid was used as a control. After inducing the expressed GFP protein into SDS-PAGE electrophoresis after incomplete denaturation, using FujiFilm (MS 3000)? In the GFP mode, the amount of protein in the GFP mode is 3 ⁇ 43 ⁇ 4.
  • the protein fibrosis was determined by Bradford method and electrophoresis scanning. The statistical results showed that the GFP protein of pGFPuv in the control group was less than 8% of the whole bacterial protein, while the high 3 ⁇ 4£4 group of p7p8 was highly efficient. GFPuv 3 ⁇ 43 ⁇ 4 GFP Protein shell U reached 25%. b) Insert the other coded three 1 ⁇ temple to do the checklist after the vector pGFPuv start codon:
  • the GlyGlySer nucleoside sequence can be replaced by the other three-fixed alcohol column, and the recombinant fluorescent protein 3 ⁇ 4 ⁇ 4 is improved by 2 to 3 times.
  • GlyGlySei ⁇ ff is listed as a difficult alcohol column substitution of SEQ ID NO: 7-21, and the corresponding 3 ⁇ 4 ⁇ pay column is SEQ ID NO: 22-31, respectively, corresponding to the following table. Even 5! — —— fiM ——— it avoids i——
  • primers P5 and p6 were engineered such that the nucleotide sequence GGGGGTTCT encoding GlyGlySer was encoded with the nucleoside Mff column GGMGCGCA (SEQ ID NO: 27) f ⁇ of GlySerAla (SEQ ID NO: 10) to form a new one.
  • the specific loading steps are as follows:
  • P9 TCTATCGATATGGGAAGOGCMTGATTACGCCAAGCTTGCA; wherein the underline is an alternate base sequence;
  • Amplification pGFPuv plasmid p3 and p4 bow I was obtained P 3p GFPuv fragment, then p9, plO bow
  • P 9pl0 was back into double-stranded DNA fragment with restriction enzymes Cla I and Hind III double digestion fragments p3p4GFPuv And the p9pl0 double-stranded DNA fragment, the restriction enzyme product is recovered and then ligated, and the ligation product is transformed into a coli ma sensation fine «, and the vine is amplified by pl, p2, and the electrophoresis tree is determined to be correct.
  • the granule I was named ⁇ 9 ⁇ 10 highly efficient "GFPuv plasmid.
  • P 9pl0 efficient " ⁇ FPuv plasmid was transferred into : Bacillus DH5 a, cultured at 37 ° C for 16 h, then ⁇ ⁇ vine, after single vine expansion culture, extract P9pl0 highly efficient " GFPuv plasmid, 3 ⁇ 43 ⁇ 4 plasmid transfer
  • the fluorescent protein was assayed in J. bacillus DH5a and the pGFPuv vector plasmid was used as a control.
  • the induced expression of the GFP protein was subjected to incompletely denaturing SDS-PAGE electrophoresis, and the amount of protein was measured by 5 ⁇ blue»3 ⁇ 4 in GFP mode using FujiFilm (LAS 3000).
  • the Bradford method and electrophoresis scanning method were used to determine the amount of peptone.
  • the statistical results showed that the GFP protein expression of pGFPuv in the control group was less than 8% of the total bacterial protein, while the high-efficiency recombination table P9pl0 was highly efficient. GFP-expressing GFP protein Then it reaches 19%.
  • SEQ ID NO: 27 Use SEQ ID NO: 27 with other nucleotides encoding 3 3 ⁇ 4 acids! ⁇ , alternative bribes are listed as SEQ ID NO: 22-26, SEQ ID NO: 28-48, and the Sfe fluorescent protein 3 ⁇ 43 ⁇ 4 ⁇ of the vine carrier has a 2 to 3 fold increase.
  • Upstream primer Pll (S ⁇ AF): TCTATCGATATGTCTTCCGGTC- TGATTACGCCAAGCnGCA, containing C/s I restriction site 3 ⁇ 43 ⁇ 4 start codon ATG, downstream bow I P12 (3 ⁇ 4ffiA-R): TGCAAGCTTGGCGTMTCATACCGGMGACATATCJGATAGA, containing ⁇ cleavage site;
  • the cytosine can be TCTTCOGGTCTG (SEQ ID NO: 50) after the original vector start codon site, and the four amino acids have 6, 6, 4, and 6 degenerate passwords. child.
  • the p3p4GFPuv fragment, and the pll and pl2 primers were annealed in the same manner as in Example 1 to form a pllpl2 double-stranded DA fragment, which was digested, ligated, and cloned to obtain a 3 ⁇ 4 ⁇ 4 initial codon and inserted into the TCTTCOGGTCTG (SEQ ID NO: 50).
  • pllpl2 efficient "Enlarged culture and extraction of GFPuv plasmid
  • the pi 1 ⁇ 12 efficient " ⁇ FPuv plasmid was transferred into Escherichia coli DH5 ⁇ , and the pi 1 ⁇ 12 high efficiency " ⁇ FPuv plasmid was extracted after expansion, and the extracted plasmid was transformed into E. coli DH5 CI into 3 ⁇ 4 ⁇ 4 fluorescent protein 3 ⁇ 4 ⁇ 4, and the pGFPuv vector was used.
  • the plasmid served as a control.
  • SDS-PAGE electrophoresis which induces incomplete denaturing of the GFP protein ship
  • the FujiFilm (LAS 3000) ⁇ ⁇ imaging system in GFP mode, ⁇ light stimulates the amount of protein in 3 ⁇ 43 ⁇ 4.
  • the Bradford method and electrophoresis scanning method were used to determine the amount of protein 3 ⁇ 4 ⁇ 4.
  • the statistical results showed that the GFP protein fibrosis of the control pGFPuv was 8% of the whole bacterial protein, while the high 3 ⁇ 4£ group of 3 ⁇ 4»body pllpl2 was highly efficient" GFPuv plasmid fiber
  • the GFP protein reached 35%.
  • the nucleic acid sequence TCTTCCGGTCTG (SEQ ID NO: 50) can also be replaced with a nucleotide sequence of AGSCTGCACTC (SEQ ID NO: 51), AGCTCTGCATTG (SEQ ID NO: 52), SerSerAlaLeu, which encodes SerSerAlaLeu.
  • AGSCTGCACTC SEQ ID NO: 51
  • AGCTCTGCATTG SEQ ID NO: 52
  • SerSerAlaLeu which encodes SerSerAlaLeu.
  • the nuclear enthalpy of primers P11 and Pl2 is 3 ⁇ 4 ⁇ 4, so that the hoof alcohol column encoding SerSerAlaLeu is SEQ ID NO: 51-52 or other sequences, respectively.
  • SerSerAlaLeu can be listed as SEQ ID NO : 53-64 S With the column, the corresponding nucleoside alcohol columns are SEQ ID NO: 65-88, respectively, and the 3 ⁇ 4f should be as follows:
  • the nuclear pi 1 and pl2 are listed as fi3 ⁇ 4i, and the nucleoside yeast TCTTCCGGTCTG (SEQ ID NO: 50) encoding SerSerAlaLeu is replaced by the nucleoside salary SEQ ID NO: 79 or 80. Book pl3, pl4.
  • the specific steps are as follows:
  • P13 TCTATOGATATG CTCAGCACACCCATGATTAOGCCAAGCTTGCA; wherein the underlined is an alternative 3 ⁇ 48 sequence; P14: TGCMGCTTGGCGTAATCATGGGTGTGCTGAGCATATCGATAGA; wherein the MS sequence underlined is ⁇ .
  • the ⁇ 13 ⁇ 14 high IH Puv plasmid was transformed into Escherichia coli DH5 a, and cultured at 37 ° C for 16 h. Then, the single sap was picked, and the single vine was expanded and cultured.
  • the Pl3pl4 high efficiency FPuv plasmid was extracted, and the extracted plasmid was transferred into Escherichia coli DH5.
  • the expression of fi3 ⁇ 4 fluorescent protein was introduced into ct, and the pGFPuv vector plasmid was used as a control. Will induce 3 ⁇ 43 ⁇ 4 ⁇ 4 GFP protein «Incompletely denatured SDS-PAGE after electrophoresis, ⁇ FujiFilm OAS 3000)? In the laparoscopic imaging system, the amount of protein in the GFP mode is 3 ⁇ 43 ⁇ 4 ⁇ 4 light.
  • the Bradford method and electrophoresis scanning method were used to determine the amount of peptone.
  • the statistical results showed that the GFP protein expression of pGFPuv in the control group was less than 8% of the total bacterial protein, while the highly efficient recombinant epigenetic group P 13pl4 was highly efficient.
  • the protein reached 34%.
  • ⁇ primer P5 (3 ⁇ 4B-F): TCTATCGATATGGCTGCATCTCTGAOGATTAOGCCMGCTTGCA, containing Cla l restriction site A start codon ATG
  • downstream primer P6 (3 ⁇ 4i positive B-R): TGCAAGCTTGGOGTAATCATAGATGCAGCCATATOGATAGA, containing Hindin restriction site.
  • a five-codon-encoded amino acid, AlaAlaSerLeuThr (SEQ ID NO: 89), which has 4, 4, 6, 6, 4, respectively, can be introduced after the original vector start codon site.
  • the degenerate codon, the nucleoside Mff of the primer encoding AlaAlaSerLeuThr is: GCTGCATCTCTGACG (SEQ ID NO: 90).
  • the results of the configuration of the 3 ⁇ 4 ⁇ 4 ⁇ 4 scoop 3 ⁇ 4 ⁇ body step m m showed that the efficiency of the recombinant "high-efficiency" GFPUV s & fluorescent protein fiber efficiency increased more than
  • AlaAlaSerLeuThr (SEQ ID NO: 89) says that it is also possible to replace with five other multi-codon amino acids.
  • the efficiency of the recombination of the high-efficiency " ⁇ 1 ⁇ fluorescent protein 3 ⁇ 4 ⁇ 4 is approximately 2 to 4 times higher.
  • Begging Buoy Primer (3 ⁇ 4i positive C-F): TCTATOGATATGGTTGTCTCGAGTGGGGGTATTACGCCMGCTTGCA, containing Cla I restriction site 3 ⁇ 43 ⁇ 4 initial codon ATG, downstream primer (3 ⁇ 4ffi C-R): TGCAAGCTTGGCGTAATCATOGAGACMCCATATOGATAGA, containing HindlllH cleavage site.
  • the sulphate can be conjugated to the six-codon-encoded S-acid ValValSer SerGlyGly (SEQ ID NO: 94), which has 4, 4, respectively.
  • ValValSerSei ⁇ lyGly (SEQ ID NO: 94) can also be replaced with another six multicodon 3 ⁇ 4 ⁇ 4 acid.
  • ⁇ for SEQ ID NO: 96" 98, the efficiency of the absolute fluorescent protein is approximately 2 to 3 times higher.
  • Begging Buoy Primer (3 ⁇ 4i positive D-F): TCTAT1XATATG CTTGTCGGCAAGCACACTCATTACGCCAAGCTTGCA, containing Cla I restriction site 3 ⁇ 43 ⁇ 4 initial codon ATG
  • downstream primer (3 ⁇ 4i positive D-R): TGCMGCTTGGOGTAATCATOGACMGAGCATATOGATAGA, containing Hindlll restriction site.
  • Seven multi-codon-encoded amino acids LeuLeuSerAlaGlyGlySer (SEQ ID NO: 99) can be introduced after the original vector start codon site, and the seven 3 ⁇ 4 acids are 6, 6, 6, 4, 4, 4, respectively.
  • LeuLeuSerAlaGlyGlySer can also be replaced with other seven multi-codon amino acids.
  • S acid For example, by replacing the S acid with the sequence SEQ ID NO: 101-103, the efficiency of the recombinant high-efficiency "GFP-GFP fluorescent protein is approximately 2-3 fold higher.
  • Example 4 Adding 10 or more multi-codon encodings to give out the 3 ⁇ 43 ⁇ 4if target gene to improve the protein 3 ⁇ 43 ⁇ 4 efficiency. a), the boat adds 10 multi-codons to the target gene to improve the efficiency of peptone
  • Trx-F 5, ⁇ GCTAGCATGTCGTTGCTCGCMGCACACTCATGAGCMCACTGTCCCA-3, underlined with A3 ⁇ 4e I restriction sites
  • Trx-R 5, -GAAnCGAAGTATTCTTTGCTGCCA-3, underlined for restriction sites
  • PCR reaction 94 ⁇ denatured 1 ⁇ 2 in, 1 cycle; 94 °C denaturation 50 s, 55 ° C annealing 50 s, 72 ° C extension lmin, 35 cycles; 72 ° C extension 10 min, 1 cycle.
  • the PCR product was electrophoresed in 1.2% of the Qiongyi, and the imaging system was photographed and the specific purpose was cut.
  • the target fragment was purified by DM-retraction recovery kit, 3 ⁇ 4A pMD18-T vector, and transformed into TOP10F' sense. Wei cells were then screened for positive clones by PCR and assayed for positive.
  • the correct positive clones were sequenced and transferred into LB liquid medium containing arginine (100 ⁇ g/mL), 200 r/min, and cultured overnight at 37 °C.
  • EZ Spin Column Plasmid Mini-Preps Kit The plasmid was extracted and 10n g plasmid was transformed into 3 ⁇ 43 ⁇ 4 host strain BL21 (DE3) pLysS sensible cells (with #tt as reference pClf T7/NT T0P0* TA Expression Kits)fect The transformation product was inserted into lOmL LB medium containing 100 ⁇ g /mL ampicillin, 34 ⁇ g/mL chloramphenicol), 200r/min, cultured overnight at 37 °C.
  • ProSerGlySerSerArgValProProThr can be replaced by SEQ ID NO: 106-107; the results indicate that the Truffle of the recombinant Trx protein has a 3 to 5 fold increase.
  • the nucleic acid sequence CCTTCT (3 ⁇ 4ATCTTCTAGAGTGCCTCCTACAGGMCA (3 ⁇ 4AGTGCTOGCMCAGCATCTCCT (SEQ ID NO: 109)) encoding a 20-codon amino acid ProSerGlySerSerArgValProProThrAlySerPro (SEQ ID NO: 108) was inserted into pCRT7 as described in the pC ® T7/NT T0P0® TA Expression Kits. 3 ⁇ 43 ⁇ 43 ⁇ 4 body, then Trx is heavy « ⁇ 3 ⁇ 43 ⁇ 43 ⁇ 4 body.
  • the facet spoon is loaded with the 7-cell bacillus T0P10F' sense-free cells, coated with LB plate containing ammonia-spermidine (100 ug/mL), cultured overnight,
  • Reverse T7 promoter (5, -TAGTTATTGCTCAGCGGT said GG-3 ') were screened for transformants. Positive gram sequencing.
  • ProSerGlySerSerArgValProProThrGlyThrGlyValLeuAlaThrAlaSerPro can be replaced by SEQ ID NO: 110-112; the amount of Trx protein is 1. 5 to 4 times higher.
  • Example 5 Ships in male cells ⁇ Adding multiple codons Difficult ethers of thrips Target genes enhancing protein «3 ⁇ 4 efficiency
  • a X-inch primer for PEGFP-C Xie sample was designed.
  • the upstream primer was (pEGFP ⁇ -S): GACATG-AGTAAAGGAGAAGAAC
  • downstream primer pEGFP-CA
  • GCGTTATTTGTAGAGCTCAT Special primer design at pEGFP ⁇
  • the 216th cleavage site Cla I of the C vector the upstream primer (pEGFP "C216-F") is: CTCATOGATATGACCATGATTACG
  • the downstream primer (pEGFP "C216-R”) is: GAGATOGATAGCTGTTTCCTGTG, both upstream and downstream, plus the CTC.
  • upstream primer F5 (high-efficiency-F): TCTATCGA- TATGGGGGGnCTATGATTAOGCCAAGCTTGCA, containing Cla I restriction site and initiation codon ATG
  • downstream primer F6 (high efficiency-R): TGCAAGCTTGGCGTAATCATA GMCCCCCCATATCGATAGA, containing Hind III cleavage site; by this primer, three polycodon-encoded acids GlyGlySer can be introduced after the original vector start codon site, and the 3 ⁇ 4 ⁇ acids have 4, 4, and 6 short codes, respectively.
  • the neuroma cells were cultured in sputum containing 10% fetal bovine serum, 1 X 12 U/L penicillin, 1 X 10 U/L streptomycin. In the middle culture, the cake was cultured at 37 °C, 53 ⁇ 43 ⁇ 4, and the fine stalks were observed regularly. The cells were subcultured with 0. 250/0 trypsin every 3 days; the neuroma was finely deleted 0 h before the transfection.
  • Lipofection reagent Lip O f eC tamine2000, GFP expression vector pEGFP "Cl, reference to method Lip O f ec tami ne 2000 transfection kit instructions, associated solution containing 5 h after transfection complex Discard, add the 10% fetal bovine serum containing the amphibian culture, continue to incubate at 37 ° C, 5 said % C cattle. Take the cell separation protein at different time points, the total protein sample obtained SDS-PAGE, it can be seen that at a good amount of about 30 kDa, the heterofluorescent protein was induced to be 3 ⁇ 43 ⁇ 4, and the amount of sputum in the highly efficient recombinant vector group was significantly higher than that of the control group.
  • GlyGlySer can also be replaced by the columns in Examples 1, 2, 3 and 4, and the experimental results show that the amount of the fluorescent protein in the pGFPuv vector is 2 to 6 times higher.
  • the method for constructing a vector of the present invention can also be used as a method for improving the efficiency of protein loading, and specifically, a nucleotide which is inserted into a cleavage sequence after ATG, which is characterized by:
  • the specific aminosaline is X1X2...Xn, wherein X represents an amino acid having 4 or more codons, and n is greater than or equal to 3. Specific examples are as follows:
  • the start codon of the SOD gene is inserted into the core of the ProSerGly (SEQ ID N0:8) to increase the efficiency of expression of the SOD protein.
  • Pro, Ser, and Gly have 6, 4, and 4 triplet codons, respectively.
  • S0D-F1 5-TAGCTAGC ⁇ 73 ⁇ 4XTAGCGGACAGCAGMGCACACC-3 (where GCTAGC % Nhe I cleavage site, ATG is the start codon; CCTAGCGGA is the inserted nuclear ⁇ column of ProSerGly) and SOD-R: 5-CGGMnCCTCTATnAGAAGCAGC -3 (GMTTC is & ⁇ / restriction site), the following cell cDNA is used as a template, and the transformation-SOD gene fragment of the S0D gene mature peptide inserted into CCTAGOGGA after PCR is amplified by the 3 ⁇ 43 ⁇ 4 initial codon.
  • S0D-F2 5'-TAGCTAGCCAGCAGAAGCACACC-3', underlined for the Nhe I restriction site
  • S0D-R 5'-CGGAATTCCTC-TATTTAGMGCAGC-3', underlined with the normal amplification of the shrimp S0D gene design, underlined For the EcoR I cleavage site, PCR is performed by cDNA amplification to obtain a normal-SOD gene fragment encoding the mature peptide of the gene.
  • the -SOD gene fragment and the normal-SOD gene fragment were respectively transformed into a PMD18-T plasmid vector, transformed into T0P10F'-sensing cells, and then positive clones were screened by PCR and sequenced 3 ⁇ 4i positive to obtain a positive clone into which the amplified fragment was inserted.
  • the positive clones were expanded and cultured, and the income and reorganization were carried out by Xie Yujian.
  • mmemwm Refer to the pCR® T7/NT TOPOB TA Expression Kits for the pC > T7/ T T0P0® TA 3 ⁇ 43 ⁇ 4 vector and the SI sequencing 3 ⁇ 4i positive 3 ⁇ 4it_S0D gene fragment and the normal-SOD gene fragment in step 1) respectively. , constructed recombinant 3 ⁇ 43 ⁇ 4
  • the constructive «3 ⁇ 4 X. faecalis T0P10F' sense-free cells were plated with LB plates containing ammoniamycin (100 ⁇ g/), cultured overnight, and positive clones containing recombinant sputum vectors were determined by sequencing with positive gram. .
  • the correct positive clones identified by 3 ⁇ 4 ⁇ sequencing were transferred to LB liquid medium containing ampicillin (100 ug/mL), 200 r/min, and cultured overnight at 37 ° C. He IJ used EZ Spin Column to describe the Plasmid Mini-
  • the plasmid was extracted from Preps Kit, and the lOng plasmid was transformed into 3 ⁇ 43 ⁇ 4 host strain BL21 (DE3) pLysS competent cells.
  • the transformed production was caged into 10 mL LB medium (containing 100 y g / nL ampicillin, 34 g/mL chloramphenicol). Incubate overnight at 37 ° C at 200 r/min.
  • the nuclear scavenger CCCAG0GGA can also be used with other cores encoding ProSerGly, as listed in SEQ ID NO: 113-140.
  • the CCCAGCGGA in the S1D-F1 and the S0D-R is respectively obtained by using the nucleotide sequence of SEQ ID NO: 113-140, and the SOD gene is subjected to the steps 1) to 3), SDS-PAGE. Electrophoresis showed a 3 to 5 fold increase in the target protein after 3 ⁇ 4it.
  • SEQ ID NO: 140 CCCAGCGGG II.
  • the nucleoside salary of the Kangma ProSe ly can be increased by 2 to 5 times.
  • nucleoside M/f column is SEQ ID NO: 22-31 0
  • the column encoding AlaLeuArg can be SEQ ID NO: 1 or SEQ ID NO: 7-21
  • the GCACTCAGA (nucleus, which can be replaced by SEQ ID NO: 2-4 or SEQ ID NO: 22-48) inserts the start codon ATG of the ⁇ -inch shrimp SOD gene to obtain the 3 ⁇ 4i-S0D gene fragment.
  • the same method was used to obtain the normal-SOD gene fragment; the ⁇ 3 ⁇ 4 SOD fragments were divided into 3 ⁇ 43 ⁇ 4A pMD18-T plasmid vector, transformed into T0P10F' sense-free cells, and then the ffi3 ⁇ 4 PCR hooves were selected for positive clones and sequenced.
  • the recombinant 3 ⁇ 43 ⁇ 4 ⁇ 4 body containing the 3 ⁇ 43 ⁇ 4_5 (» and normal-SOD gene fragments was constructed by the method of Example 1).
  • the constructed bacillus bacillus T0P10F' sense-free cells were plated, coated with LB plates containing ammoniamycin (100 ⁇ g/), cultured overnight, and positively cloned to determine positive clones containing recombinant «3 ⁇ 4 vector. .
  • the recombinant host strain containing the 3 ⁇ 4it_S0D and normal-SOD gene fragments was induced by IPTG, and the 3D 43 ⁇ 44 line analysis of the target protein was carried out by SDS-PAGE analysis.
  • Example 7 Inserting a gene encoding the four 4 ⁇ sedation » acid after the start codon of the gene of interest to improve protein efficiency
  • SerLeuGlyArg nucleus enhances the 3 ⁇ 4 ⁇ 4 efficiency of SOD protein.
  • Ser, Leu, Gly, and Arg have 6, 6, 4, and 6 codons, respectively.
  • RNA was prepared from shrimp blood cells and reverse transcribed into cD A.
  • Wo 1 J uses a silk I S0D-F1 designed according to the shrimp SOD gene: 5-TAGCTAGCATGAGCCT0GGAAGACAGCAG- AAGCACACC-3
  • the GCTAGC is a Nhe / restriction site, ATG is the start codon;
  • AGCCT0GGAAGA is the inserted nucleic acid sequence encoding SerLeuGlyArg);
  • S0D-R 5- OGGMTTCCTCTATTTAGMGCAGC-3 (GMTTC is & ⁇ /? / restriction site),
  • the cDNA was amplified by PCR to obtain a gene fragment of the 3 ⁇ 48-derived mature peptide inserted into the core»column AGCCTCGGAAGA after the start codon.
  • Wo uses the normal amplified pre-primer SOD-F2 (5'-TAGCTAGCCAGCAGAAGCACACC-3, underlined as Nhe I restriction site) and S0D-R designed according to the shrimp SOD gene.
  • the PCR product was electrophoresed in 1.2% of the Qiong earning, and the nucleus was photographed, and the specific target DNA was cut and recovered.
  • the purified target fragment was ligated into the PM to express the D18-T plasmid vector.
  • T0P10F' competent cells were then screened for positive clones and tested positively.
  • the recombinant expression vector was constructed by following the instructions in the pCR® T7/NT T0P0B TA Expression Kits, the plasmid containing the pC > T7/ T TPO0® TA expression vector and the recovered gene fragment containing the SOD gene mature peptide.
  • the constructed expression vector was transformed into E. coli T0P10F' sense-free cells, and the L-book B plate containing ampicillin (100 y & mL) was coated and cultured overnight. ⁇ Positive gram ⁇ -sequence was determined to contain recombinant «3 ⁇ 43 ⁇ 4 body Positive clone.
  • the positive clones identified by ⁇ ! sequencing were transferred to LB liquid medium containing ammonia (100 g/mL) and cultured overnight. ⁇ I" using EZ Spin Column Plasmid Mini-Preps Kit
  • the transformed product was introduced into 10 mL of LB medium (containing 100 w g/nL ampicillin, 34 u g/mL chloramphenicol), 200 r/min, and cultured overnight at 37 °C.
  • the amount of recombinant protein constructed by the post-insertion sequencer accounted for 40% of the total amount of the total bacterial protein, while the amount of the recombinant protein constructed using only the original sequence containing the SOD accounted for only 13% of the amount of the bacterial protein.
  • the code T3 ⁇ 4rArgProAla (the SMi ⁇ column can be replaced by SEQ ID N0: 53 ⁇ 64) is paid by ACMGACC0GCA (this The nucleic acid sequence corresponding to SEQ ID NO: 65-88 can be inserted into the start codon ATG of the shrimp SOD gene to prepare shrimp blood cell RNA and reverse transcribed into cDNA.
  • Wo 1 J uses primer S0D-F1 designed according to the shrimp SOD gene: 5- TAGCTAGCATGH I ⁇ m ⁇ /CAGCAGMGCACACC-3 (the obliquely underlined core lli column can be replaced by SEQ ID NO: 65-88); SOD- : 5 ⁇ 0GGMnCCTCTATTTAGMGCAGC- 3 (GAAHC is EcoR / restriction site), m ⁇ PCR amplification of cDNA was obtained into lj3 ⁇ 4 ⁇ codon and inserted into the nucleus, and the gene fragment of the mature peptide of AGCCT0GGMGA was listed.
  • the normal primer S0D-F2 (5, -TAGCTAGCCAGCAGMGCACACC-3 ', underlined as Nhe I restriction site) and S0D-R (5, -QGGMTTCCTCTATT TAGAAGCAGC-3', underlined using the normal amplified primer designed according to the shrimp SOD gene
  • S0D-F2 5, -TAGCTAGCCAGCAGMGCACACC-3 ', underlined as Nhe I restriction site
  • S0D-R 5, -QGGMTTCCTCTATT TAGAAGCAGC-3', underlined using the normal amplified primer designed according to the shrimp SOD gene
  • the site was digested with EcoR I.
  • the cDNA was amplified by cDNA and a gene fragment encoding the mature peptide of the gene was obtained.
  • PCR reaction M/3 ⁇ 4 Denaturation at 94 °C 1 ⁇ 2iin, 1 cycle; Denaturation at 94 °C for 50s, annealing at 55 °C for 50s, extension at 72°C for 1min, 35 cycles; extension at 72°C for 10min, 1 cycle.
  • the PCR product was electrophoresed in 1.2% of the Qiongyue sputum, and the Sl-class imaging system was photographed and then cut for specific purposes.
  • the target fragment was purified by DNA-crossing recovery kit, 3 ⁇ 4A pMD18-T plasmid vector, and transformed into TOP10F' Competent cells were then screened for positive clones by PCR and tested for miscellaneous. Book
  • the recombinant expression vector was constructed by following the instructions in the pCR® T7/NT T0P0® TA Expression Kits, the pC > T7/ T T0P0® TA expression vector and the recovered gene fragment containing the SOD gene mature peptide.
  • the constructed expression vector was transformed into E. coli T0P10F' sensitized cells, and LB plate containing ampicillin (100 ug/mL) was applied and cultured overnight. The positive gram was tested with the recombinant sputum. Positive clone.
  • the correct positive clones identified by ⁇ ! sequencing were transferred to LB liquid medium containing aminomycin (100 ⁇ g/niL), cultured at 200 r/min, 37 V overnight.
  • LB liquid medium containing aminomycin (100 ⁇ g/niL)
  • pLysS sense-free cells with lateral reference pC > T7/NT T0P0® TA Expression Kits.
  • the transformed product was introduced into 10 mL of LB medium (containing 100 w/mL ammonia, penicillin, 34 g/mL chloramphenicol), 00r/min, 37. C was cultured overnight.
  • the sample was treated with 500 y L cracking night, and repeatedly frozen and thawed in liquid nitrogen and 42 ° C water bath.
  • the SJ-PAGE analysis of the target protein by SDS-PAGE showed that the amount of the recombinant protein constructed by inserting the A sequence with the start codon was 3 ⁇ 4 ⁇ The total amount of the total protein was 26 Between % and 48, the recombinant protein constructed with only the original sequence of SOD was used as a 3 ⁇ 4 ⁇ 4 ⁇ 4% of the amount of the protein.
  • the reagents and materials of the present invention bow kiss synthesized by Shanghai Bioengineering Co., Ltd.; PCR cymbal pMD18_T, restriction Endonuclease, Taq enzyme, Lianjun were purchased from Bao Bioengineering Co., Ltd., IPTG, etc. were purchased from Shanghai Bioengineering Co., Ltd., and the original nuclear 3 ⁇ 43 ⁇ 4# pCR® T7/NT TOPO® TA and pEGFP, pGFPuv were purchased from Clontech. ⁇ coli DH5 a Roots Biochemical Technology (Beijing) Co., Ltd.
  • J uses the method of the advancement of the gene peptone efficiency of the present invention, and the target gene can be efficiently realized by simply performing the target gene or vector, and the vector with high efficiency characteristics can be constructed.
  • the method of the invention can increase the efficiency of the protein of any gene of interest by more than 2 times, or the carrier of the existing protein 3 ⁇ 4 ⁇ 4 carrier, thereby increasing the efficiency of the protein 3 ⁇ 4 ⁇ 4 by more than 2 times.
  • a simple and easy-to-use method will play an important role in the regulation of genes, protein clocks, and so on.

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Abstract

La présente invention concerne un vecteur d'expression pour augmenter l'efficacité d'expression de protéine. Une séquence nucléotidique spécifique est introduite en aval du codon d'initiation ATG du vecteur d'expression, et code pour au moins 3 résidus d'acide aminé. Ces résidus d'acide aminé correspondent à 4 codons ou plus. La présente invention concerne en outre un procédé pour augmenter l'efficacité d'expression de protéine. Le procédé de la présente invention peut être utilisé dans la modification d'un gène quelconque pour augmenter son efficacité d'expression. Par rapport à l'art antérieur, le vecteur d'expression ou procédé de la présente invention peuvent augmenter l'efficacité d'expression de protéine cible de 2 à 5 fois.
PCT/CN2011/071030 2011-02-16 2011-02-16 Procédé pour augmenter l'efficacité d'expression de protéine et vecteur d'expression à cet effet Ceased WO2012109788A1 (fr)

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CN105886525A (zh) * 2016-06-25 2016-08-24 吉林大学 高表达外源蛋白的毕赤酵母株
CN109750036B (zh) * 2017-11-03 2022-07-12 普莱柯生物工程股份有限公司 核苷酸序列、使用其提高蛋白表达效率的方法及应用
CN111850028A (zh) * 2020-08-10 2020-10-30 武汉恒意赛生物科技有限公司 一种提高蛋白表达效率的方法
CN119592593A (zh) * 2024-12-12 2025-03-11 武汉伯远生物科技有限公司 一种提高蛋白表达量的方法

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