ABSTRACT
Objective To knockout and identify the Antigen 43 (Ag43) in the Escherichia Coli JM109 .Methods Mutation group Ⅱ introns RNA protein complexes (RNP) gene sequence was obtained by Sigma Company′s TargeTron Gene Knockout Sys‐tem and Ag43 gene specific designed PCR primers amplification ,then ,to acquired Ag43 specific recombinant RNP plasmid pACD4K‐Ag4 ,this gene sequence was inserted into the plasmid pACD4K‐C of RNA′s expression .Finally ,pEGFP‐Ag43 was trans‐formed into JM109 and inserted the group Ⅱ intron into the Ag43′s locus by IPTG inducing expression .Results The best insertion locus was between 1 812 and 1 913 .Through the agarose electrophoresis gel ,the RNP gene sequence was consistent with the expec‐ted value (350 bp) .The pEGFP‐Ag43 vector was correctly constructed which was proofed by endonuclease Nhe Ⅰ and Hind ⅡI di‐gestion as predicted products (3 646 and 4 029 bp;7 000 and 550 bp ,respectively ) .The PCR and gene sequence results indicated that the group Ⅱ intron was inserted into the locus between 1 812 and 1 913 in the Ag43 gene .Conclusion Successful knockout of the Ag43 in Escherichia Coli JM109 found basis to further study the Ag43′s function and regard the coli as host bacteria of Ag43 chimeric protein recombinant .
ABSTRACT
In order to investigate the anti-tumor angiogenesis activity with a recombinant Ag43/FGFR1 chimeric protein(AF)vaccine in a mouse H22 hepatoma model,tumor volume and survival rate of the mice were studied at a 3-day interval,Microvessel density(MVD)was detected by immunohistochemistry.The endothelial deposition of autoantibodies within tumor tissues was examined by immunofluorescent staining,and anti-FGFR1 antibody-producing B cells(APBCs)were tested by enzyme-linked immunospot(ELISPOT)assay.Compared with the three control groups,the tumor volume was significantly decreased and the survival time was significantly prolonged in AF-immunized group(P<0.05).The number of APBCs in AF-immunized mice(129.6±10.9)was more than in controls[6.2±1.1(FGFR1),6.0±1.2(Ag43)and 5.2±1.4(NS),P<0.01].Moreover,the endothelial deposition of autoantibodies was found in tumor tissues from AF-immunized mice,but not in control groups.MVD in AF-immunized group was significantly lower than in FGFR1-immunized group,Ag43-immunized group and NS group(10.3±3.1 vs 39.4±8.6 vs 42.3±9.8 and 43.6±10.6,P<0.01).These findings demonstrated that the AF protein vaccine effectively inhibited tumor angiogenesis and growth via production of autoantibodies against self-FGFR1.