Plasmid vector capable of implementing efficient cloning and temperature-induced expression and construction method of plasmid vector

A plasmid carrier and construction method technology, applied in the field of molecular biology, can solve the problems of increased screening pressure, false positives, and low cloning efficiency, and achieve the effects of avoiding restriction, flexible and diverse cloning methods, and convenient protein purification

Inactive Publication Date: 2018-08-03
SHENYANG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the common disadvantage of existing plasmid vectors is that the cloning efficiency is not high, and false positives often occur during the cloning process, thereby increasing the pressure of subsequent screening

Method used

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  • Plasmid vector capable of implementing efficient cloning and temperature-induced expression and construction method of plasmid vector
  • Plasmid vector capable of implementing efficient cloning and temperature-induced expression and construction method of plasmid vector
  • Plasmid vector capable of implementing efficient cloning and temperature-induced expression and construction method of plasmid vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1: the acquisition of plasmid vector pANY3

[0039] 1) Use a pair of primers PHis-for1 (5'-AAAAC TGCAG CACCA CCACC ACCAC CACTA AGGCTGCTAA CAAAG CCCGA AAGGA AGCTG-3') and PHis-rev1 (5'-GTAGT TTATC ACAGT TAAAT TGCTAACGCA GTCAG GGATA TCCGG ATATA GTTCC TCCTT TC-3 '), the plasmid pET3a was used as a template for PCR amplification, and the amplified product was named Fragment 1. The reaction conditions for PCR amplification are: 10 μmol / L each primer, 2U Pfu enzyme, 1x Pfubuffer, 0.2mmol / L dNTPs, about 2ng template, and add sterilized deionized water to 20μl. The cycle program of PCR amplification was: pre-denaturation at 94°C for 2 min, followed by 25 cycles of denaturation at 94°C for 30 s, annealing at 63°C for 30 s and extension at 72°C for 2 min. The reaction was extended at 72°C for 10 min before the end of the reaction.

[0040]2) Using a pair of primers PET9-for1 (5'-GAAAG GAGGA ACTAT ATCCG GATAT CCCTG ACTGCGTTAG CAATT TAACT GTGAT AAACT AC-3') and PHis-re...

Embodiment 2

[0053] Example 2: Cloning of the endo-β-1,3-glucanase pfLamA gene into pANY3

[0054] 1) The plasmid pANY3 was digested with AhdI, and the enzyme digestion reaction system was as follows: 120 μl of enzyme digestion system contained 10 U of AhdI endonuclease (NEB Company), 4 μg of pANY3 plasmid DNA, and 1× buffer. After enzyme digestion at 37°C for 6 hours, the large fragment after enzyme digestion is the T vector, named pANY3-T, which does not need to be recovered by gel.

[0055]2) Use pfLamA-For-AhdI (5'-GGCAT GGTCC CTGAA GTGAT AGAAA TAGAT GGAAAACAG-3') and pfLamA-Rev-AhdI (5'-AGGAC CACTA ACGAA TGAGT AAACC CTTAC ATAAT CC-3') as forward and reverse primers, The plasmid pET9d-pfLamA (Ilari A, Fiorillo A, Angelaccio S, Florio R, Chiaraluce R, van der Oost J, Consalvi V. Crystal structure of a family 16endoglucanase from the hyperthermophile Pyrococcus furiosus--structural basis of substrate recognition. FEBS J. 2009; 276(4):1048-1058.) as a template for PCR amplification. The...

Embodiment 3

[0060] Embodiment 3: Trichoderma reesei endoxylanase Hj-Xyn gene is cloned into pANY3

[0061] 1) Using Trichoderma reesei cDNA as template, Hj-Xyn-NdeI-For(5'-GGAGG TAAAA CATAT GACGATCCAA CCAGG CACGG GCTAC AACAA CGG-3') and Hj-Xyn-PstI-Rev(5'-AAAAC TGCAGGCTAACGGTG ATGCT TGCAGAACCG CTGCT G-3') as forward and reverse primers for PCR amplification. The reaction conditions for PCR amplification are: 10 μmol / L each primer, 2U Pfu enzyme, 1x Pfu buffer, 0.2mmol / L dNTPs, about 2ng template, add sterilized deionized water to 20μl. The cycle program of PCR amplification was: pre-denaturation at 94°C for 2 min, followed by 25 cycles of denaturation at 94°C for 30 s, annealing at 63°C for 30 s and extension at 72°C for 3 min. The reaction was extended at 72°C for 10 min before the end of the reaction.

[0062]2) Digest the fragment obtained in the above step 1 and pANY3 with NdeI and PstI, perform 1% agarose gel electrophoresis and gel recovery and purification, and connect with T4 DN...

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Abstract

The invention relates to the field of genetic engineering, and in particular to a plasmid vector, which is capable of flexibly implementing non-background TA cloning, cohesive end cloning and blunt end cloning and is capable of implementing temperature-induced protein expression, and a construction method of the plasmid vector. The plasmid vector is closed-ring shaped double-stranded DNA, which contains a gene, which is capable of coding toxic protein ccdB, as a negative selection marker. A flexible cloning function and a temperature-induced expression function of the plasmid are verified. Theplasmid, which is more convenient and efficient, can play an important role in the field of genetic engineering.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a plasmid vector capable of flexibly performing background-free TA cloning, sticky-end cloning and blunt-end cloning, and capable of temperature-induced protein expression and a construction method thereof. Background technique [0002] Plasmids can be used not only for molecular cloning, but also as vectors carrying target genes for protein expression. At present, there are some expression vectors that can realize high-efficiency protein expression in E. coli, including pET (StudierFW, Rosenberg AH, Dunn JJ, Dubendorff JW: Use of T7RNA polymerase to direct expression of cloned genes. Methods Enzymol 1990,185:60 –89.), pTrc (Amann E, OchsB, Abel KJ: Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli. Gene 1988,69(2):301–315.) and pBAD (Guzman LM , Belin D, Carson MJ, Beckwith J: Tight regulation, modulation, and hig...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/66
CPCC12N15/70C12N2830/34
Inventor 安迎锋高何瑞高嵩王虹玲许淑敏张艺峰刘霞刘一菲张铮泽哈基李秀通廉兴隆
Owner SHENYANG AGRI UNIV
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