Precise quantification method of lentivirus packaging helper plasmids
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A technology of lentivirus packaging and auxiliary plasmids, which can be used in biochemical equipment and methods, microbial measurement/testing, etc., and can solve problems such as poor accuracy and repeatability
Inactive Publication Date: 2017-12-05
安徽安龙基因科技有限公司
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[0004] The purpose of the present invention is to overcome the defects of poor accuracy and repeatability
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Embodiment 1
[0028] A method for accurately quantifying lentiviral packaging helper plasmids, comprising the steps of:
[0029] (1) Design specific primers for the special genes (gag gene, rev gene and vsv-g gene) carried by the three auxiliary plasmids respectively
[0030] (2) Detection of lentiviral packaging helper plasmid
[0031] S1 uses PCR to amplify the corresponding fragments of each gene;
[0032] S2 constructs each gene product on the T vector to form a new cloning plasmid;
[0033] S3 transforms the cloned plasmid into E. coli, identifies, cultures, and extracts the plasmid;
[0034] S4 Calculate the corresponding copy number according to the nucleic acid concentration of the three plasmids and the molecular weight of the recombinant plasmid, and use this as a standard;
[0035] Among them, the copy number calculation formula is: copy number = 6.02 × 1023 × nucleic acid concentration ÷ (DNAlength × 660); the unit of copy number is copies / ml, and the unit of nucleic acid con...
Embodiment 2
[0040] A method for accurately quantifying lentiviral packaging helper plasmids, comprising the steps of:
[0041] (1) Design specific primers for the special genes (gag gene, rev gene and vsv-g gene) carried by the three auxiliary plasmids respectively, and according to the lentiviral gene sequence (GenBank: D86068. Design a pair of specific primers for the vsv-g gene, as shown in the following table:
[0042]
[0043] (2) Detection of lentiviral packaging helper plasmid
[0044] S1 Amplify the corresponding fragments of each gene with ordinary PCR: The PCR reaction system is as follows: use three auxiliary plasmids as templates, and add them to new PCR tubes respectively; add 2 μl of corresponding upstream and downstream primers to the PCR tube; then Add 4 μl of dNTP, 5 μl of 10×PCR Buffer in sequence, and add to 50 μl with sterile water; finally add 0.25 μl of high-fidelity DNA polymerase.
[0045] Among them, the PCR reaction conditions are: pre-denaturation 95°C / 2min...
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Abstract
The invention discloses a precise quantification method of lentivirus packaging helper plasmids, comprising design of specific primers for special genes respectively carried by three helper plasmids and detection of lentivirus packaging helper plasmids. The special genes are gag gene, rev gene and vsv-g gene, and a pair of specific primers is respectively designed for the gag gene, rev gene and vsv-g gene. According to a traditional method, concentration of extracted plasmids is detected, and mole number is calculated on the basis of molecular weight of plasmids. The traditional method is simple and feasible, but cannot achieve precise quantification of packaging plasmids and has poor repeatability in a virus packaging optimization experiment. In comparison with the prior art, the invention is to design the specific primers for the special genes respectively carried by the three helper plasmids and carry out precise quantification of packaging helper plasmids by fluorescent quantitative PCR. Through experiments, specific copy number of the three packaging helper plasmids can be known. Thus, data support is provided for subsequent optimization of plasmid ratio, and experimental accuracy and repeatability are raised.
Description
technical field [0001] The invention relates to the technical field of lentiviral packaging, in particular to a method for accurately quantifying lentiviral packaging helper plasmids. Background technique [0002] Lentivirus vector is a gene therapy vector developed on the basis of HIV-1 (Human Immunodeficiency Virus Type 1), which has a wide spectrum of infection, can effectively infect dividing and quiescent cells, and long-term stable expression of foreign genes, etc. Advantages, so it becomes a powerful tool for introducing foreign genes. [0003] Lentiviral packaging systems are generally three-plasmid or four-plasmid packaging systems. Since the four-plasmid system is more biosafety than the three-plasmid system, it is widely used. Among them, in addition to the transfer plasmid in the four-plasmid expression system, the other three packaging auxiliary plasmids each carry some special genes, namely gag gene, rev gene and vsv-g gene. In order to improve the efficienc...
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