Packaging method for rapidly obtaining high-titer lentivirus

Abstract

The invention discloses a packaging method for rapidly obtaining high-titer lentivirus, which comprises the following steps: mixing lentivirus packaging plasmids, a target gene expression plasmid, a transfection reagent and protamine to form liposome, wherein the lentivirus packaging plasmids are pRsv-REV, pMDLG-pRRE and pVSV-G; adding the formed liposome into tool cells for transfection and culture; and collecting the cultured virus supernatant, and treating to obtain a virus solution. Through the mode, the optimized and improved formula of the four-plasmid system of the lentivirus packaging system and the transfection reagent are adopted, the virus packaging period can be greatly shortened, the time cost is saved, the method does not need to repeatedly replace medium for cells, the packaging steps are simple and easy to operate, the types of reagents used in the method are reduced, the operation is easy, and the lentivirus packaged by the method is high in titer and high in purity, and can be used for animal-level research.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a packaging method for rapidly obtaining high-titer lentivirus. Background technique [0002] Lentivirus is a type of retrovirus with a double-stranded RNA genome. However, unlike general retroviruses, lentiviruses have a wider host range and have the ability to infect both dividing and non-dividing cells. After the lentiviral genome enters the cell, it is reverse-transcribed into DNA in the cytoplasm to form a pre-DNA integration complex. After entering the nucleus, the DNA is integrated into the cell genome. The integrated DNA is transcribed into mRNA, returned to the cytoplasm, expresses the target protein, or produces small RNA; the recombinant lentiviral vector is based on HIV-1 (human immunodeficiency virus type I), and is developed using PAX and P2G coat proteins The raised tool vector, whose virulence gene has been deleted and replaced by an exogenous target gene, is a pseu...

AI Technical Summary

Problems solved by technology

[0009] (1) The current lentivirus packaging system is cumbersome and difficult

General-level laboratories cannot obtain virus tools by themselves, and can only obtain lentiviral particles with the help of professional institutions

However, the time period is very long. It usually takes one month from the time of obtaining the lentiviral plasmid to the completion of the lentiviral packaging. The time is long and the efficiency is low, which seriously affects the progress of researchers' experiments.

[0010] (2) At present, there are many steps for lentivirus packaging: a. It is necessary to replace the complete medium of the tool cells with a serum-free medium before lentivirus packaging. The serum-free medium will inevitably affect the growth and state of the tool cells. After transfection The poor condition directly leads to low production, and the virus titer cannot meet the follow-up experiments; b. 8 hours after liposome transfection, the serum-free medium needs to be replaced with complete medium

Frequent liquid changes will cause the tool cells that are not firmly attached to the wall to fall off, thereby affecting the packaging effect; c, the next 24 hours, 48 ​​hours, 72 hours or even 96 hours must carefully absorb the supernatant and replace with fresh medium. It all floats up, resulting in wasted efforts; d. Subsequent supernatants need to be centrifuged, filtered, purified, concentrated, etc., which are more cumbersome, and the lentiviral particles can be collected and titered before use

The whole process has strict requirements on cell growth, technical operation and human judgment, and the steps are cumbersome and complicated. If one step is wrong, it may be necessary to start from scratch. Moreover, the time requirements for researchers are relatively strict. Researchers usually work overtime to wait for the time point of the experiment. Once it is done, it cannot be stagnated in the middle, and the experiment cycle is longer, which seriously affects the efficiency of the experiment

[0011] (3) At present, the lentivirus packaging liposome configuration system is complicated and there are many reagents. For example, using lipo3000 transfection method, the liposome preparation process is very cumbersome, and it is necessary to add a specific medium without serum and double antibodies, and then calculate the quality of each packaging plasmid of the virus , the amount of lentiviral vector target plasmid, lipo3000 and P3000, transfection reagents and plasmids should be separated and incubated in different EP tubes, and then added and mixed one by one, like the four-plasmid packaging system needs to absorb, add and mix operations before and after 10 More than one time, and there is a certain order, the process is very cumbersome, if you are not careful, you will add the wrong one, it is easy to miss or add the wrong reagent

[0012] (4) Although there are many types of lentiviral vectors on the market, the expression intensity of the fluorescent genes carried by them is weak, and the infection efficiency cannot be judged intuitively

There are many types of lentiviral vectors available, and it takes a lot of time and energy to select the vectors. Once the wrong choice is made, it will affect the experimental results

Moreover, the expression intensity of the fluorescent gene carried by the existing lentiviral vector is relatively weak. After the lentivirus infects the cells, the fluorescence observed under the fluorescence microscope within 48 hours is very weak, and the infection efficiency cannot be accurately judged. Cytometer to detect infection efficiency brings a lot of inconvenience to researchers

[0013] (5) At present, the titer of lentiviruses on the market is generally low, and the approximate titer is 1E+8TU / mL. This titer is only suitable for infecting common susceptible tumor cells. For primary cells, nerve cells, and stem cells that are difficult to infect , endothelial cells, cardiomyocytes, etc., need to be repeatedly infected to obtain a weak effect, and even after repeated infection, the infection still fails

[0014] (6) Due to factors such as low titer and poor purity, lentiviruses currently on the market can only be used for cell-level infection, while for animal-level experiments, high virus titer and high purity are required

Many researchers have to do both in vitro cell level research and in vivo animal level research, so they have to package lentivirus and adeno-associated virus at the same time, which greatly increases the cost of scientific research

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