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Intracellular fusion expression type pre-T vector, preparation and application

A technology of internal fusion and vector, which is applied in the field of intracellular fusion expression type pre-T vector, can solve the problems of negative cloning and connection, and achieve the effect of rapid construction

Inactive Publication Date: 2009-03-11
ZHEJIANG UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Second, if the spatial positions of the two XcmI restriction sites are relatively close, the restriction efficiency of the carrier will be affected. Even if the purity of the carrier DNA is high and the quality of the enzyme is also good, there will be a "knife" of the restriction carrier. , this linear vector cannot be ligated with PCR products, but the vector is self-ligated, resulting in the generation of negative clones

Method used

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  • Intracellular fusion expression type pre-T vector, preparation and application
  • Intracellular fusion expression type pre-T vector, preparation and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1: LacI gene site-directed mutation eliminates XcmI site

[0053] Using the SOE method, using pET39 as a PCR template, the six primers were designed as follows:

[0054] Lac-1: 5'-ttaggatccgcgacccatttgct-3' (including BamHI site)

[0055] Lac-2: 5'-agagatatccgcaccaacgcgca-3' (including EcoRV site)

[0056] Lac-3: 5'-ctgattggcgttgctacctccagtctggccctgca-3' (for mutation site 1)

[0057] Lac-4: 5'-gccagactggaggtagcaacgccaatcagcaacga-3' (for mutation site 1)

[0058] Lac-5: 5'-tcccactgcgatgttagttgctaacgatcagatggcgct-3' (for mutation sites 2 and 3)

[0059] Lac-6:5'-catctgatcgttagcaactaacatcgcagtgggaacgatgc3' (for mutation sites 2 and 3)

[0060] Table 2: Sequence comparison before and after site mutation

[0061] Original sequence (with XcmI recognition site) New sequence (no XcmI recognition site) bit

point

1 Gccacctccagtctggcc

GCC ACC TCC AGT CTG GCC

Ala Thr Ser Ser Leu Ala

gcTacctccagtctggcc

GCT ACC TCC AG...

Embodiment 2

[0074] Embodiment 2: the preparation of XcmI restriction cassette and the construction of front T carrier

[0075] Primers A and B serve as templates for PCR1 reaction:

[0076] A: 5′-ATACTTAAGCGAGAAAAAAGGTTCTGGTTCTGGTCAT-3′

[0077] B: 5′-GACATTAACCTATCCATTAGATCCATGGTGATGATGATGATGACCAGAACCAGAACC-3′

[0078] The reaction parameters were as follows: denaturation at 94°C for 10 min, annealing at 48°C for 300 s, and extension at 72°C for 10 min.

[0079] With pET11b as template, utilize following two primers to carry out PCR2 reaction:

[0080] Amp1: 5′-CACCATGGATCTAATGGATAGGTTAATGTCATGATAATA-3′

[0081] Amp2:5′-CTTAAGCTTCCAAGGGTATAATGGAAATGAAGTTTTAAATCAATC-3′

[0082] The reaction parameters were as follows: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 120 s, 30 cycles, and extension at 72°C for 10 min.

[0083]The above two PCR1 and PCR2 product fragments were used for gene splicing reaction with prime...

Embodiment 3

[0085] Embodiment 3: the removal of DsbA signal peptide gene (keep ATG as initiation codon):

[0086] Design primers:

[0087] DsbA-1:5'-AAG CATATG GCGCAGTATGAAGAT-3', (the underlined part is NdeI recognition sequence)

[0088] DsbA-2:5'-TCG CTTAAG TATTTCACTGT-3', (the underlined part is the recognition sequence of BspTI)

[0089] PCR template: pET39 plasmid (or Escherichia coli genomic DNA is also available, pET39 plasmid is recommended as a template)

[0090] PCR parameters: 94°C for 3min

[0091] 94℃ 30s 40℃ 30s 72℃ 1min 3 cycles

[0092] 94℃ 30s 48℃ 30s 72℃ 1min 27 cycles

[0093] 72°C 10min

[0094] 4°C 10min

[0095] Digest the PCR product and pET39-2 with NdeI and BspTI, replace the original DsbA gene in pET39-2 with the DNA fragment without the signal peptide gene, and obtain a recombinant plasmid without the DsbA signal peptide gene, that is, the intracellular fusion expression type T vector creates conditions for realizing t...

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Abstract

The invention provides a cell fusion expression pre-T vector, which is prepared by the following method: firstly, a DNA1 fragment of BspTI-GSGSG-HHHHHH-XcmI-Amp-XcmI-HindIII is established and encoded; secondly, the DNA sequence of three XcmI sites in an LacI gene in a site-directed mutation pET39 plasmid is changed under the condition that the encoding amino acid sequence is unchanged, so as to lose XcmI recognition sites, and site-directed mutation pET39-1 which eliminates the XcmI sites is obtained; thirdly, DNA1 is directionally recombined into pET39-2 by utilization of BspTI and HindIII; and fourthly, a DsbA signal peptide gene in the pET39-2 is removed, and the cell fusion expression pre-T vector is obtained. Besides the XcmI for preparing a linear T vector, the invention does not need to use other restriction enzymes, can further establish fusion expression genetic engineering bacteria in one step, and provides a feasible technical method for preparing a plurality of bioactive peptide engineering bacterium libraries.

Description

(1) Technical field [0001] The invention relates to an intracellular fusion expression type pre-T vector which can be used for rapidly constructing an engineering bacterium for intracellular fusion expression of a target gene and its preparation and application. (2) Background technology [0002] Using Escherichia coli to prepare biologically active peptides with medicinal value is the most commonly used technical route for genetic engineering, but the target gene expression product is easy to form inclusion bodies when overexpressed, and the first amino acid is generally methionine, which is different from natural biological activity. The amino acid sequence of the peptide is inconsistent, and it is easy to produce immunogenicity when used as a medicine. It is necessary to explore the purification process, and it is difficult to express small molecular weight peptides. [0003] The fusion protein expression strategy can be used to solve the above problems well: after the f...

Claims

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Application Information

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IPC IPC(8): C12N15/63
Inventor 林陈水于真真杨丹燕许明任慧颖
Owner ZHEJIANG UNIV OF TECH
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