[Construction and expression of HSV-2gD-Hsp70 fusion protein gene].
To construct and express Hsp70-HSV2gD fusion protein. Genes of Hsp70 and HSV-2gD were subcloned into vectors pGEX-4T-1 respectively. After confirmed by DNA sequence analysis, the recombinant plasmids pGEX-4T-HSP-gD was transformed into E. coli DH5alpha and induced to express with IPTG. The expressed protein was characterized by SDS-PAGE and Western blot after purified. BALB/c mice were immunized with fusion proteins respectively via intra-m uscular injection. The proliferation of spleen lymphocytes, the level of y-IFN in culture and anti-HSV-2gD IgG antibody in serum was detected was detected. The expressed protein was analyzed by SDS-PAGE after induced with IPTG, which showed a new band with an apparent molecular mass corresponding to the predicted size (118 kD). Western Blotting analysis demonstrates that the purified Hsp70-HSV2gD fusion protein had specific binding activity. The stimulation indexes of spleen lymphocytes, the level of gamma-IFN in culture and anti-HSV-2gD IgG antibody in serum of GST-Hsp70-gD group was obviously higher than that of other groups (P < 0.05 respectively). The successful expression of the Hsp70-HSV2gD fusion protein, which can induce immune responses, laid a solid foundation for its further research.
Fan
Jian-Yong
JY
Department of Dermatology, Guangzhou General Hospital, Guangzhou 510010, China. fanjianyong@hotmail.com
Yang
Hui-Lan
HL
Wang
Ying
Y
Guan
Lei
L
D000818:Animals; D015153:Blotting, Western; D049109:Cell Proliferation; D015870:Gene Expression; D018840:HSP70 Heat-Shock Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D007074:Immunoglobulin G / Q000097:blood / Q000276:immunology; D007371:Interferon-gamma / Q000097:blood; D051379:Mice; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D013154:Spleen / Q000166:cytology / Q000276:immunology; D014759:Viral Envelope Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification
D018840:HSP70 Heat-Shock Proteins; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C040570:glycoprotein D-herpes simplex virus type 2; D007371:Interferon-gamma