ABSTRACT
Objective To analyze the differential expression of D2 and D3 alternative splicing sites of human kinectin in human hepatocellular carcinoma (HCC) tissues, adjacent non-cancerous tissues and normal liver tissues, and to investigate a possible relationship between alternative splicing sites of kinectin and hepatocarcinogenesis. Methods The cDNA was obtained by RT-PCR in 45 coupled HCC cancerous and adjacent tissues, and 10 normal liver tissues. The difference in the expression of D2 and D3 alternative splicing sites in cDNA was examined by semi-quantitative PCR, and statistical analysis was performed. Results The ratio of D2L (long segment contains of the D2 region)/D2S (short segment that does not contain a D2 zone) in hepatocellular cancerous tissues was 2.709 ± 1.025, the ratio of D2L/D2S in adjacent non-cancerous tissues was 1.564 ± 0.357, and the ratio of D2L/D2S in normal liver tissues was 1.507 ± 0.499. The differences were statistically significant (F=29.698, P 0.05). Conclusion Variant containing D2 is over expressed in cancerous tissues and this alteration may be tumor associated.
ABSTRACT
Objective:Have disclosed that the Behcet's patient have autoantibodies to 160 kD protein which is subsequently identified as protein kinectin by gene library screening using a Behcet's patient serum(BD4).To investigate the antigeneic location of kinectin.Methods:Purified RNA from Hep2 line culture and amplified three kinectin fragment by RT PCR using three pairs of primers(kin 3,base sequence 920～ 1 346;kin M,503～994;kin 5,22～510),which combine to cover nearly the full sequence of kinectin molecules listed in Genbank(Z22551);then cloned the three fragments into pET 42a(+) vector and expressed in BL21(DE3) E.coli.The whole cell lysate was put onto SDS PAGE and subsequently transferred to a nitrocellous membrane,then detected for the antibody of Behcet's patient serum by ECL system.Results:Three PCR fragment posed a 99% correspondent rate with kinectin sequence.All of the expression vectors has a correct readframe and expressed three fusion peptides of molecular weight 89(kin 5)?89(kin M) and 82 kD(kin 3) respectivey by Western blot analysis.Of eight patients,6 patients serum reacted to kin M,5 to kin 3 and 1 to kin 5;none of ten normal controls reacted to all the three fusion fragments.Conclusion:Three PCR fragment of kinectin covering sequence 133 to 4 107(aa22～1 346) have been successfully cloned into pET 42a(+) vector and expressed in BL21(DE3) E.coli.The preliminary analysis demonstrates that the antigeneic region of kinectin is mainly located in the middle and carboxyl terminal portion.