Objective： The aim of the study was to observe the transfection efficacy of hepatitis B virus envelope （HBVE） and evaluate its ability as a gene transfer vector for liver cancer cells. Methods： To obtain HBVE, the...Objective： The aim of the study was to observe the transfection efficacy of hepatitis B virus envelope （HBVE） and evaluate its ability as a gene transfer vector for liver cancer cells. Methods： To obtain HBVE, the supematant fluid of HepG 2.2.15 cells was mixed with a PEG8000 solution for concentration and was inactivated by β-propiolactone. The acquired HBVE was used to pack plRES2-EGFP to test its package ability. Then, we examined its quantity and quality with ELISA, PCR, SDS-PAGE and electron microscopy. The plRES2-EGFP was packed with HBVE and obtained the product HBVE-GFP. The plRES2-EGFP was packed with liposome and obtained the product liposome-GFP. HBVE-GFP and liposome-GFP were used to transfer HepG 2 cells to study the transfection efficiency. HBVE-GFP was used to transfer HepG 2, A549, HeLa and FB cells to study the targeting ability. The green fluorescent protein （GFP） expression was observed under a fluorescent microscope. The rate of GFP positive cells was determined by flow cytometry. Results： 1. The acquired HBVE could retain the surface protein HBsAg ＋ pre S1 ＋ pre S2 and had no virus DNA. It had good package ability for plRES2-EGFP. 2. Transfection efficiency： The GFP could be observed in both the liposome group and HBVE group under the fluorescent microscope. But the HBVE group had a higher fluorescent intensity than liposome group. The transfection rate of liposome group was 49.97% ＋ 2.37% while the HBVE group was 70.65% ＋ 3.15% and the fluorescent intensity of the HBVE group was 3-4 times （P = 0.000） for liposome group with the determination of flow cytometry. 3. Targeting ability： The GFP could be observed in the four groups under the fluorescent microscope. The HepG 2 group had the highest fluorescent intensity among the four groups. The transfection rate of HepG 2 group was 71.35% ＋ 0.03% which was highly expressed than other groups （P = 0.000） and the fluorescent intensity of the HepG 2 group was 2-3 times （P = 0.000） for the other 3 groups with the determination of flow cytometry. Conclusion： HBVE can be constructed successfully with the methods of PEG8000 and β-propiolactone from the supernatant fluid of HepG 22.15 cells. The HBVE can be a candidate gene transfer vector for liver cancer cells.展开更多
The envelope gene gp85 of ev/J, a new family of endogenous avian retroviral sequences identified recently, has the most extensive nucleotide sequence identity ever described with ALV-J avian leukosis virus. This repor...The envelope gene gp85 of ev/J, a new family of endogenous avian retroviral sequences identified recently, has the most extensive nucleotide sequence identity ever described with ALV-J avian leukosis virus. This report described expression of ev/J envelope gene gp85 derived from commercial meat-type chicken using the Invitrogen Bac-to-Bac baculovirus expression system. The antigenicity and immunoreactivity of the recombinant endogenous gp85 gene product (SU) were analyzed by indirect immunofluorescence, Western blot, indirect and blocking Enzyme-Linked ImmunoSorbent Assay (ELISA) using JE9 monoclonal antibody (MAb) against the envelope protein of ALV-J (ADOL-4817), positive mouse antiserum against the ev/J gp85 SU and sera from chicken naturally infected with ALV-J. The results showed that the ev/J gp85 SU can bind specifically to JE9 MAb and antiserum from chicken naturally infected with ALV-J, and the binding reactivity between exogenous ALV-J gp85 SU and natural positive chicken serum against exogenous ALV-J can be blocked by positive mouse serum against the ev/J gp85 SU. It is concluded that recombinant endogenous gp85 gene product (SU) has close immunological relatedness to the envelope protein of exogenous ALV-J (ADOL-4817 and IMC10200 strain).展开更多
犬瘟热病是由犬瘟热病毒 (CDV)感染犬和其他食肉动物的多发性、致死性传染病。CDV属于副黏病毒科麻疹病毒属有囊膜的负链线性RNA病毒 ,基因组长约 16kb。基因组中包括 6个非重叠基因区。它们的编码基因按 3′～ 5′端顺序依次为N P M F ...犬瘟热病是由犬瘟热病毒 (CDV)感染犬和其他食肉动物的多发性、致死性传染病。CDV属于副黏病毒科麻疹病毒属有囊膜的负链线性RNA病毒 ,基因组长约 16kb。基因组中包括 6个非重叠基因区。它们的编码基因按 3′～ 5′端顺序依次为N P M F H L系列。最新研究表明CDV两个糖蛋白 (H ,F)是宿主免疫系统的主要目的抗原 ,产生中和抗体的重要抗原之一。N蛋白是保守性较强的免疫原性蛋白 ,此外N蛋白上含有T细胞表位。H 。展开更多
基金Supported by a grant from the National Natural Sciences Foundation of China No 30100189
文摘Objective： The aim of the study was to observe the transfection efficacy of hepatitis B virus envelope （HBVE） and evaluate its ability as a gene transfer vector for liver cancer cells. Methods： To obtain HBVE, the supematant fluid of HepG 2.2.15 cells was mixed with a PEG8000 solution for concentration and was inactivated by β-propiolactone. The acquired HBVE was used to pack plRES2-EGFP to test its package ability. Then, we examined its quantity and quality with ELISA, PCR, SDS-PAGE and electron microscopy. The plRES2-EGFP was packed with HBVE and obtained the product HBVE-GFP. The plRES2-EGFP was packed with liposome and obtained the product liposome-GFP. HBVE-GFP and liposome-GFP were used to transfer HepG 2 cells to study the transfection efficiency. HBVE-GFP was used to transfer HepG 2, A549, HeLa and FB cells to study the targeting ability. The green fluorescent protein （GFP） expression was observed under a fluorescent microscope. The rate of GFP positive cells was determined by flow cytometry. Results： 1. The acquired HBVE could retain the surface protein HBsAg ＋ pre S1 ＋ pre S2 and had no virus DNA. It had good package ability for plRES2-EGFP. 2. Transfection efficiency： The GFP could be observed in both the liposome group and HBVE group under the fluorescent microscope. But the HBVE group had a higher fluorescent intensity than liposome group. The transfection rate of liposome group was 49.97% ＋ 2.37% while the HBVE group was 70.65% ＋ 3.15% and the fluorescent intensity of the HBVE group was 3-4 times （P = 0.000） for liposome group with the determination of flow cytometry. 3. Targeting ability： The GFP could be observed in the four groups under the fluorescent microscope. The HepG 2 group had the highest fluorescent intensity among the four groups. The transfection rate of HepG 2 group was 71.35% ＋ 0.03% which was highly expressed than other groups （P = 0.000） and the fluorescent intensity of the HepG 2 group was 2-3 times （P = 0.000） for the other 3 groups with the determination of flow cytometry. Conclusion： HBVE can be constructed successfully with the methods of PEG8000 and β-propiolactone from the supernatant fluid of HepG 22.15 cells. The HBVE can be a candidate gene transfer vector for liver cancer cells.
基金Natural Science Foundation of China (30460098)China Postdoctoral Science Foundation funded project (2005038585)
文摘The envelope gene gp85 of ev/J, a new family of endogenous avian retroviral sequences identified recently, has the most extensive nucleotide sequence identity ever described with ALV-J avian leukosis virus. This report described expression of ev/J envelope gene gp85 derived from commercial meat-type chicken using the Invitrogen Bac-to-Bac baculovirus expression system. The antigenicity and immunoreactivity of the recombinant endogenous gp85 gene product (SU) were analyzed by indirect immunofluorescence, Western blot, indirect and blocking Enzyme-Linked ImmunoSorbent Assay (ELISA) using JE9 monoclonal antibody (MAb) against the envelope protein of ALV-J (ADOL-4817), positive mouse antiserum against the ev/J gp85 SU and sera from chicken naturally infected with ALV-J. The results showed that the ev/J gp85 SU can bind specifically to JE9 MAb and antiserum from chicken naturally infected with ALV-J, and the binding reactivity between exogenous ALV-J gp85 SU and natural positive chicken serum against exogenous ALV-J can be blocked by positive mouse serum against the ev/J gp85 SU. It is concluded that recombinant endogenous gp85 gene product (SU) has close immunological relatedness to the envelope protein of exogenous ALV-J (ADOL-4817 and IMC10200 strain).
文摘犬瘟热病是由犬瘟热病毒 (CDV)感染犬和其他食肉动物的多发性、致死性传染病。CDV属于副黏病毒科麻疹病毒属有囊膜的负链线性RNA病毒 ,基因组长约 16kb。基因组中包括 6个非重叠基因区。它们的编码基因按 3′～ 5′端顺序依次为N P M F H L系列。最新研究表明CDV两个糖蛋白 (H ,F)是宿主免疫系统的主要目的抗原 ,产生中和抗体的重要抗原之一。N蛋白是保守性较强的免疫原性蛋白 ,此外N蛋白上含有T细胞表位。H 。
