A kind of engineering cell line and its construction method and application

A cell line and engineering technology, applied in the field of insect engineering cell line and its establishment, can solve the problems of time-consuming, laborious, inability to obtain baculovirus titer, low ratio, etc.

Active Publication Date: 2017-02-15
武汉金益肽生物有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the process of detecting the titer of recombinant baculovirus, AcMNPV is not very sensitive to the cell line of Spodoptera frugiperda (Sf9 for short), so a more realistic baculovirus titer cannot be obtained
Therefore, the use of Sf9 cell line to detect the titer of recombinant baculovirus is not accurate.
[0007] At the same time, due to the very low ratio of recombinant baculoviruses (usually only 0.1%-1%), the traditional plaque screening technology is time-consuming and laborious to screen recombinant baculoviruses, and even skilled researchers need 1-2 months to complete. Complete, and the purity of the obtained virus particles is not high

Method used

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  • A kind of engineering cell line and its construction method and application
  • A kind of engineering cell line and its construction method and application
  • A kind of engineering cell line and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Embodiment 1: (as figure 1 shown, taking the Tn5ET&P cell line as an example)

[0086] S10, obtaining the sequentially connected exogenous DNA fragment 603+PHp+GFP+HygB+IE1p+1629, the nucleotide sequence of the exogenous DNA fragment is shown in SEQ ID NO.7.

[0087] S20, the above-mentioned exogenous DNA fragments were gel-recovered and ligated with the transfer plasmid pBlueScript II SK(+), introduced into Top10 cells, and identified by enzyme digestion to obtain a recombinant plasmid containing 603+PHp+GFP+HygB+IE1p+1629 DNA sequence , the BlueBac-GFP / HYG recombinant plasmid. Wherein, the nucleotide sequence of pBlueScript II SK(+) is shown in SEQ ID NO.6, and the nucleotide sequence of BlueBac-GFP / HYG recombinant plasmid is shown in SEQ ID NO.8.

[0088] S30, take 8 μl Gene Juice transfection reagent, add it to 200 μl insect serum-free medium, mix and let stand for 5 minutes, add 4 μg DNA of the BlueBac-GFP / HYG recombinant plasmid obtained in step S20, mix well an...

Embodiment 2

[0091] Embodiment 2: (taking Tn5ET&P cell line as example)

[0092] S10, the recombinant baculovirus solution was continuously diluted 10 times in a centrifuge tube, starting from 10 -5 diluted to 10 -12 , for later use; dilute the Tn5ET&P cell suspension to 5×10 with culture medium 3 pcs / hole, spare.

[0093] S20, take 50 μl of the diluted recombinant baculovirus solution and inoculate it into a 96-well cell culture plate containing 100 μl of Tn5ET&P cell suspension, and inoculate one longitudinal row for each dilution, a total of 8 wells; Base 50 μl, inoculated into a porous cell culture plate containing 100 μl of Tn5ET&P cell suspension, as a control group; the inoculated Tn5ET&P cell suspension was cultured in an incubator, and the results were observed and recorded every day until the fifth day.

[0094] S30, according to the expression of the reporter gene, count the number of infected wells, the number of uninfected wells, the number of all infected and uninfected we...

Embodiment 3

[0106] Embodiment 3: Contrast Tn5ET&P cell line and Sf9 cell line containing gfp (hereinafter referred to as Sf9ET&P cell line)

[0107] The engineering cell line Sf9ET&P was obtained with reference to the method described in Example 1, and the recombinant baculovirus titer was detected according to the method described in Example 2. The test results are shown in Tables 3 and 4 below:

[0108] Table 3 Statistical results of recombinant baculovirus infection of Sf9ET&P cells

[0109] Dilution infected hole uninfected hole 10 -5

12 0 10 -6

12 0 10 -7

9 3 10 -8

2 10 10 -9

0 12 10 -10

0 12 10 -11

0 12 10 -12

0 12 control group 0 12 control group 0 12

[0110] Table 4: Calculation of infection rate of Sf9ET&P cells

[0111] Dilution number of infections Uninfected infection rate 10 -6

23 0 100.0％ 10 -7

11 3 78.6％ 10 -8

2 13 13.3％ ...

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Abstract

The invention relates to an engineering cell line. The engineering cell line is a cabbage looper Tn5 cell line, and the cabbage looper Tn5 cell line carries a reporter gene which can perform expression when a cell is infected with baculovirus. With the adoption of the engineering cell line, when the Tn5 cell is infected with the recombinant baculovirus, once the recombinant baculovirus replicates in the Tn5 cell, the Tn5 cell can start the expression of the reporter gene, and a reporter protein is generated; by monitoring the reporter protein, the infection condition of the recombinant baculovirus to the cell can be more intuitively observed.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to an insect engineering cell line and its establishment method and application. Background technique [0002] Baculovirus Expression Vector System (hereinafter referred to as BEVS) is a recombinant protein expression system that uses a recombinant baculovirus carrying an exogenous target gene as a vector to express in insects or their cultured cells. [0003] Compared with other expression systems, this system can obtain high-level expression of recombinant protein, and the highest expression level of recombinant protein can reach 50% of the total protein of cells. The obtained recombinant protein has complete biological functions, and can complete complete post-translational processing in BEVS, including glycosylation, phosphorylation, acylation, signal peptide excision, peptide cleavage and decomposition, etc., modified sites It is completely consistent with the situation of n...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N15/85C12Q1/70C12R1/93
Inventor 谭业平李晓峰孙彦涛袁丽汤军芝
Owner 武汉金益肽生物有限公司
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