CN-122012416-A - Method for improving slow virus titer

Abstract

The invention relates to the technical field of virus utilization, and particularly provides a method for improving slow virus titer, which is used for screening a compound used in a production process, the concentration of the compound and the harvesting time. On one hand, the mechanism is beneficial to reducing the reinfection of the produced lentivirus BaEVless on production cells and improving the virus titer, and on the other hand, in the production process of the invention, heparin sodium can obviously reduce apoptosis of the production cells after fusion, increase the survival time of the cells and increase the virus yield.

Inventors

• SU YANXIN
• ZHENG CHENGYUN
• LIU ZHICHAO
• Pang Qingxiao
• LI JINXUE
• JIANG YANG
• ZHANG HONGJIA
• LUO SHENGTAO

Assignees

• 山东丽山生物科技有限公司
• 济南启辰达尔生物技术有限公司

Dates

Publication Date

20260512

Application Date

20260414

Claims (6)

1. 1. A method for improving lentivirus titer is characterized in that, the method for improving the slow virus titer comprises the following steps: Step one, cell plating is carried out on cells according to the cell density of (2-2.5) multiplied by 10 5 cell/cm 2 , and the cells are placed into a 37 ℃ incubator for culture; Carrying out plasmid transfection by using plasmids after 24 hours, wherein the dosage of a transfection agent PEI is 3 times that of the plasmids, and the plasmids comprise pCDH-GFP plasmid, pLP1 plasmid, pLP2 plasmid and BaEVless plasmid, wherein the total dosage of the plasmids is 0.22-0.35 mug/cm 2 , and the mass ratio of each plasmid is 6:4:4:1; And thirdly, adding heparin sodium into a culture dish when the liquid is changed after the plasmid is transfected for 6 hours, so that the final concentration of the culture medium is 10-75U/mL, and taking the supernatant after the plasmid is transfected for 48-72 hours to obtain the available lentivirus.
2. 2. The method of claim 1, wherein the step-cell density is 2.2x10 5 cell/cm 2 .
3. 3. The method of claim 1, wherein the total amount of step two is 0.3 μg/cm 2 .
4. 4. The method for improving lentivirus titer according to claim 1, wherein the step of heparin sodium is 10-25 u/mL.
5. 5. The method of claim 1, wherein the step of increasing the titer of the lentivirus is 10U/mL.
6. 6. The method of claim 1, wherein the supernatant is obtained 48 hours after transfection of the plasmid in step three.

Description

Method for improving slow virus titer Technical Field The invention relates to the technical field of virus utilization, in particular to a method for improving slow virus titer. Background Lentivirus BaEVless can efficiently infect dividing cells and non-dividing cells, is a vector widely used in gene editing, and lentivirus vector BaEVless-LV is a key production raw material of cell therapies such as CAR-T, CAR-NK. The third generation lentivirus vector is usually a four-plasmid system, comprising a shuttle plasmid carrying a target gene and pLP1, wherein the shuttle plasmid can provide virus core structural protein and replicase Gag/Pol and pLP2, and a helper plasmid containing Rev, can promote the nuclear output and efficient packaging of virus RNA, and BaEV-Rless-LV or VSV-G-LV, has the functions of respectively expressing BaEV-gp or VSV-G envelope glycoprotein, realizing virus pseudotyping and determining a host cell infection spectrum, and can obviously reduce virus recombination risk and improve biosafety and vector consistency by separating a virus replication essential gene env from the structural gene, and finally generate the available virus particles by co-transfecting the plasmids into 293T production cell lines. The virus particles can be used for transduction immune cells for targeted cell therapy, but the infection efficiency of traditional vectors VSV-G-LV is low for difficult transduction cells such as NK cells and gamma delta T cells, and the BaEV-Rless-LV is more efficient than the traditional vectors VSV-G-LV, but the titer is low and the vector is difficult to use. The slow virus titer is low, firstly, after deleting R peptide sequence, fusion activity is enhanced, 293T cells are abnormally fused in the production process to form syncytia, and shedding apoptosis after cell fusion obviously reduces virus yield, secondly, a're-infection' effect is commonly existed in slow virus production, namely, newly generated virus particles can reenter 293T cells to generate non-replicative progeny viruses lacking gag/pol/env and other key genes, so that the titer of the available viruses is diluted. Thus, there is a need to develop a method of increasing lentiviral titer that inhibits syncytial shedding apoptosis and reduces viral reinfection. Disclosure of Invention In order to solve the problems, the invention aims to provide a method for improving lentivirus titer. The method for improving the slow virus titer comprises the following steps: Step one, cell plating is carried out on cells according to the cell density of (2-2.5) multiplied by 10 5cell/cm2, and the cells are placed into a 37 ℃ incubator for culture; Carrying out plasmid transfection by using plasmids after 24 hours, wherein the dosage of a transfection agent PEI is 3 times that of the plasmids, and the plasmids comprise pCDH-GFP plasmid, pLP1 plasmid, pLP2 plasmid and BaEVless plasmid, wherein the total dosage of the plasmids is 0.22-0.35 mug/cm 2, and the mass ratio of each plasmid is 6:4:4:1; And thirdly, adding heparin sodium into a culture dish when the liquid is changed after the plasmid is transfected for 6 hours, so that the final concentration of the culture medium is 10-75U/mL, and taking the supernatant after the plasmid is transfected for 48-72 hours to obtain the available lentivirus. Further, the step one cell density was 2.2X10 5cell/cm2. Further, the total amount of the second step is 0.3 mug/cm 2. Further, the sodium heparin in the step is 10-25U/mL. Further, the sodium heparin in the step (A) is 10U/mL. Further, the supernatant was taken 48 hours after the plasmid transfection in step three. The invention has the following beneficial effects: Heparin Sodium serves as a high-sulfated glycosaminoglycan, and the molecular structure of Heparin Sodium contains dense sulfonic acid groups and carboxylic acid groups, so that the Heparin Sodium has high negative charge density. In the invention, the binding can effectively shield positive charges on the surfaces of virus particles, thereby weakening unnecessary electrostatic adsorption between viruses and production cell membranes and strengthening non-heparan sulfate HS dependence. On one hand, the mechanism is beneficial to reducing the reinfection of the produced lentivirus BaEVless on production cells and improving the virus titer, and on the other hand, heparin sodium can obviously reduce apoptosis after fusion of the production cells in the production process, increase the cell survival time and increase the virus yield. It should be noted that, from experimental results, the optimal implementation parameters of the invention can raise the crude virus titer of the lentivirus BaEVless to 1.1X10 7 TU/mL, and the lentivirus BaEVless produced by using the optimal implementation parameters can efficiently infect NK cells and gamma delta T cells with the infection efficiency of more than 80%. Drawings The drawings described herein are included to provide a further understa