Preparation of scFv-LAAO fusion protein and its anti-proliferative effect on human lung adenocarcinoma H460 cells and mouse lung adenocarcinoma cells
YANG Guangyong
GUO Haitao
LIU Qianming
HE Guangzhi
Abstract：Objective To prepare the fusion protein of recombinant anti-interleukin-4 receptor (IL-4R)single anti-body gene and Trimeresurus stejnegeri venom L-amino acid oxidase (LAAO)gene (scFv-LAAO fusion protein),and then to observed its anti-proliferative effect on human lung adenocarcinoma H460 cells and mouse pulmonary adenocarcinoma cells.Methods ① We connected IL-4R scFv gene and LAAO gene to make the scFv-LAAO fusion gene by splicing o-verlapping extension PCR (SOE-PCR),constructed the pET28a-scFv-LAAO recombinant plasmid,and then used the plas-mid to transfect the BL21(DE3)prokaryotic expression bacterium and induced it to express the scFv-LAAO fusion protein. The molecular weight of scFv-LAAO fusion protein was detected by SDS-PAGE,and the expression of His-tag of scFv-LAAO fusion protein was detected by Western blotting.② Setting the control group,cyclophosphamide group and fusion protein group (including high-dose group,medium-dose group and low-dose group),and in the six groups we separately cultured the human lung adenocarcinoma cells H460 with cell culture medium,cyclophosphamide (2.0 mg/mL),and scFv-LAAO fusion protein (4.0,2.0 and 1.0 mg/mL).We detected and calculated the inhibition rates of cell prolifera-tion of the cyclophosphamide group and fusion protein group (high-dose group,medium-dose group and low-dose group)by MTT.Seventy-five mice were selected to establish the animal models of lung adenocarcinoma,and then were randomly di-vided into the control group,cyclophosphamide group and fusion protein group (including high-dose group,medium-dose group and low-dose group),and the six groups were separately given physiological saline,cyclophosphamide (0.1 mL/10g),scFv-LAAO fusion protein (100,50 and 20 mg/kg)through intraperitoneal injection once per day for 10 days.Sev-en days after drug withdrawal,we weighed the mice of each group,calculated the anti-tumor rate of cyclophosphamide group,high-dose group,medium-dose group and low-dose group of fusion protein,at the same time we recorded the surviv-al time of statistics.Results The length of scFv-LAAO fusion gene was 2 000~3 000 bp.After the prokaryotic expression bacterium BL21(DE3)was transfected by the fusion gene,the fusion protein scFv-LAAO was extracted from the superna-tant of bacteria lysate,and its molecular weight was about 86 kD,which coincided with scFv-LAAO fusion protein,and the His-tag of scFv-LAAO fusion protein was detected by Western bloting.No significant difference was found in the cell prolif-eration inhibition rate,anti-tumor rate and survival time of human lung adenocarcinoma H460 cells between the cyclophos-phamide group and high-dose fusion protein group (all P>0.05 ).The cell proliferation inhibition rate,anti-tumor rate and survival time of human lung adenocarcinoma H460 cells of the cyclophosphamide group and high-dose fusion protein group was higher than that of the medium-dose and low-dose fusion protein groups (all P<0.05 ).Meanwhile,the cell proliferation inhibition rate,anti-tumor rate and survival time of the low-dose group was lower than that of the medium-dose group (all P<0.05).Conclusion The scFv-LAAO fusion protein is successfully prepared,and ScFv-LAAO fusion pro-tein can inhibit the proliferation of human lung adenocarcinoma H460 cells and mouse lung adenocarcinoma cells.
Keywords：anti-interleukin-4 receptor single antibodyTrimeresurus stejnegeri venom L-amino acid oxidaselung neoplasmspulmonary adenocarcinomacell proliferation
Publication Date：2016-01-01
Online Publishing Date：2025-08-15(First online date of this platform, not the publication date of the document)
Pages：5( 4-8 )