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Cloning and expression method of Klebsiella pneumniae nifH gene

A Klebsiella, expression method technology, applied in the field of cloning and expression of nifH gene, can solve the problem that nifH gene cannot be expressed

Inactive Publication Date: 2013-01-09
HEILONGJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although it is known that termite intestinal nifH genes have high diversity, only a small number of nifH genes can be preferentially transcribed and expressed, and most nifH genes cannot be expressed

Method used

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  • Cloning and expression method of Klebsiella pneumniae nifH gene
  • Cloning and expression method of Klebsiella pneumniae nifH gene
  • Cloning and expression method of Klebsiella pneumniae nifH gene

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Experimental program
Comparison scheme
Effect test

specific Embodiment approach 1

[0017] Specific embodiment one: the cloning and expression method of Klebsiella pneumoniae nifH gene in this embodiment are carried out according to the following steps:

[0018] 1. Take 3mL of Klebsiella pneumoniae HUB-IV-004 bacterial liquid, centrifuge at 12000r / min for 1min, discard the supernatant, and then extract genomic DNA according to the operation method of the bacterial genomic DNA extraction kit of TIANGEN company;

[0019] 2. Perform PCR amplification using the genomic DNA extracted in step 1 as a template to obtain the target gene nifH, and then connect it to the pMD18-T vector to obtain the recombinant plasmid pMD18-T-nifH;

[0020] 3. Extract the recombinant plasmids pMD18-T-nifH and pET28a plasmids according to the operation method of the TIANGEN plasmid mini-extraction kit, and then use EcoR I and Nde I to perform double enzyme digestion on the recombinant plasmids pMD18-T-nifH and pET28a plasmids respectively, and recover all Need the target fragment, conne...

specific Embodiment approach 2

[0033] Specific embodiment two: the difference between this embodiment and specific embodiment one is that the reaction system of PCR amplification in step two is as follows:

[0034]

[0035] PCR amplification conditions were pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 1 min, a total of 35 cycles; final extension at 72°C for 10 min; and incubation at 4°C. Other steps and parameters are the same as those in Embodiment 1.

specific Embodiment approach 3

[0036] Specific embodiment three: the difference between this embodiment and specific embodiment one is that the system of double enzyme digestion in step three is as follows:

[0037]

[0038] Other steps and parameters are the same as those in Embodiment 1.

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Abstract

The invention provides a cloning and expression method of a Klebsiella pneumniae nifH gene and relates to a cloning and expression method of a nifH gene. The method comprises the following steps: extracting a genome DNA (deoxyribounucleic acid) of Klebsiella pneumniae; carrying out PCR (polymerase chain reaction) amplification so as to obtain a target gene nifH, connecting the target gene nifH with a vector so as to obtain a recombinant plasmid Pmd18-T-nifH; extracting the plasmid, carrying out double-enzyme digestion, constructing a recombinant plasmid PET28a-nifH, transforming the recombinant plasmid PET28a-nifH to Escherichia coli so as to obtain Escherichia coli containing PET28a-nifH; then inoculating the Escherichia coli containing PET28a-nifH to an LB (Luria-Bertani) liquid culturemedium containing kanamycin for culture to obtain a culture bacterial liquid; and adding the culture bacterial liquid to the LB liquid culture medium containing kanamycin for culture, then adding (isopropyl beta-D-1-thiogalactopyranoside) IPTG for culture and carrying out inducing expression, wherein the inducing expression is finished if specific strips occur through SDS-PAGE (sodium dodecyl sulfonate-Polyacrylamide gel electrophoresis) analysis. The method provided by the invention has an important theory basis and a reality sense for developing termite-killing biological medicament which is not toxic to people and livestock and is environmentally-friendly.

Description

technical field [0001] The invention relates to a method for cloning and expressing nifH gene. Background technique [0002] Termites belong to the order Arthropoda, Insecta, Insecta, Isoptera, and are relatively ancient social pests. The economic losses caused by termite damage to buildings and crops amount to more than 100 billion U.S. dollars every year. Existing medicaments for killing termites are all chemical medicaments, so they all have very large toxic and side effects and cause serious environmental pollution. The use of biotechnology to develop termite biological control agents with high efficiency, non-toxicity, long residual effect and little impact on the environment has become the main direction of termite control agents in the future. [0003] Termite is a typical oligotrophic organism, and the nitrogen fixation of intestinal nitrogen-fixing microorganisms is the main source of nitrogen in termites. At present, scientists have cloned a large number of nifH...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/31C12N15/10C12N15/70C12R1/22
Inventor 赵凯张小燕
Owner HEILONGJIANG UNIV