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Plasmid standard substance containing human thymalfasin target gene as well as preparation method and application of plasmid standard substance

A thymus method and standard product technology, which is applied in the field of detection of plasmid copy number in recombinant human thymus method bacteria, can solve the problem of inability to accurately quantify the plasmid copy number of recombinant human thymus method bacteria, and achieves good repeatability. Effect

Inactive Publication Date: 2018-03-23
HUNAN FANGSHENG PHARMACEUTICAL CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] The purpose of the present invention is to provide a kind of plasmid standard product containing the new target gene of human thymus method, its preparation method and its application in the detection method and the test kit of plasmid copy number in recombinant human thymus method new engineering bacteria, to solve the present problem The problem of the inability to accurately quantify the copy number of the plasmid in the newly engineered bacteria of the recombinant human thymus method in the existing technology

Method used

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  • Plasmid standard substance containing human thymalfasin target gene as well as preparation method and application of plasmid standard substance
  • Plasmid standard substance containing human thymalfasin target gene as well as preparation method and application of plasmid standard substance
  • Plasmid standard substance containing human thymalfasin target gene as well as preparation method and application of plasmid standard substance

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preparation example Construction

[0025] 3. Preparation of plasmid standards

[0026] The plasmid standard product of the present invention is a pET plasmid vector containing a specific nucleotide sequence inserted into the new target gene of the human thymus method. The vector can be amplified in Escherichia coli and prepared by using a plasmid extraction kit.

[0027] 4. Synthetic specific primers

[0028] In one embodiment, the primers for amplifying the new target gene of the human thymus method are specifically:

[0029] Upstream primer as shown in SEQ ID No: 2: 5'-CACAACTCTGATGCTGCTGT-3'

[0030] Downstream primer as shown in SEQ ID No: 3: 5'-ATCTCCCGTGATGCAGTTCT-3'.

[0031] 5. Using real-time fluorescent quantitative PCR to detect the copy number of recombinant human thymus method new engineered bacteria plasmid

[0032] (1) preparing a plasmid standard product containing the new target gene of the human thymus method, the plasmid standard product contains the Ssp-Ta1-Mxe gene sequence;

[0033](2)...

Embodiment 1

[0054] Embodiment 1: the preparation method of the plasmid standard product containing the new target gene of human thymus method

[0055] (1) Clone the target gene into the cloning vector: synthesize the new target gene of human thymus method shown in SEQ ID No: 1, and connect it to the cloning vector pUC57;

[0056] (2) Double enzyme digestion reaction: The cloning plasmid vector pUC57-Ssp-Ta1-Mxe containing the target gene and the expression plasmid vector pET-28a were digested according to the enzyme digestion systems shown in Table 1 and Table 2 respectively, using Nco I and Xho I carried out the double enzyme digestion reaction respectively.

[0057] Table 1: pUC57-Ssp-Ta1-Mxe double enzyme digestion system

[0058]

[0059] Table 2: pET-28a double enzyme digestion system

[0060]

[0061] Band recovery: Perform electrophoresis detection on the digested products of the double enzyme digestion reaction, and recover the bands with a size of about 5200bp in the dige...

Embodiment 2

[0065] Embodiment 2: Determination of recombinant E. coli plasmid copy number

[0066] Pick a recombinant Escherichia coli colony and inoculate it into 5ml of LB liquid medium (containing 100μg / m1 kanamycin), culture at 37°C and 220rpm for 3 hours (OD is about 2), take 200μl of centrifuged bacteria, wash with water Once, the thalline was taken by centrifugation, dissolved in 1ml of water, lysed at 95°C for 10 minutes, and diluted 10 times with water as the test solution.

[0067] Use Roche’s FastStart Essential DNA Green Master reagent, target gene amplification primers shown in SEQ ID No: 2 and SEQ ID No: 3, and water to prepare PCR premix reaction solution 1 to contain the new target gene of human thymus method The plasmid standard is used to build a standard curve for the template, such as figure 2 shown.

[0068] The PCR premix reaction solution 2 was prepared with Roche’s FastStart Essential DNA Green Master reagent, Escherichia coli single-copy gene primers and water,...

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Abstract

The invention provides a plasmid standard substance containing a human thymalfasin target gene as well as a preparation method and application of the plasmid standard substance. The plasmid standard substance is plasmid containing an Ssp-Ta1-Mxe gene sequence. According to the plasmid standard substance, the problem that in the prior art, a plasmid copy number in recombinant human thymalfasin engineering bacteria cannot be accurately quantified so that proper engineering bacteria are difficult to screen can be solved.

Description

technical field [0001] The invention relates to the field of recombinant protein engineering bacteria screening, in particular to a plasmid standard product containing a new target gene of the human thymus method, its preparation method and a detection method and kit for the plasmid copy number in the recombinant human thymus method new engineering bacteria in the application. Background technique [0002] In the process of studying the expression of bioengineered recombinant proteins, the plasmid copy number is a parameter that needs to be considered, and its accurate quantification is very important. Generally speaking, the expression level of the recombinant protein increases with the increase of the plasmid copy number, but when the copy number is large, the relationship between the copy number and the expression level of the recombinant protein does not exist. There are two reasons: on the one hand, the expression level of exogenous recombinant protein is no longer det...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/66C12N15/12C12Q1/689
CPCC07K14/57581C12N15/66C12N15/70C12Q1/689C12Q2600/166
Inventor 张庆华陈波夏红英王峥
Owner HUNAN FANGSHENG PHARMACEUTICAL CO LTD
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