Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition · 2007
[Gene fragments cloned and immune recognition studied preliminarily for desmoglein 4 in pemphigus vulgaris].
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Li Wei, Feng Yun, Lu Xiao-yan, Li Jing-yi, Ran Yu-ping
Аннотация
OBJECTIVE: To amplify the nucleotide sequences of desmoglein 4 (Dsg4) extracellular domains (EC1-4) from human skin tissue, and then to investigate their roles in pemphigus vulgans (PV) pathogenesis.
METHODS: RNA was obtained from normal human skin tissue and then cDNA was synthesized by RT-PCR. The target gene fragments of desmoglein 4 extracellular domains (EC1-4) were amplified by PCR. With the technique of gene recombination, these target gene fragments were inserted into pET32a plasmids respectively by T4 DNA ligase, which formed the recombinant plasmids used to transform the E. coli DH5alpha competent germs. Screening of transformant germs was done by LB medium with Ampicillin. The DNA sequences of positive recombinants were then identified. The epitopes of four recombinant proteins of Dsg4 in PV patients were analyzed by ELISA.
RESULTS: Four DNA bands with all the length of 350 bp were obtained by RT-PCR. Consequently four expression plasmids of desmoglein 4 extracellular domains were constructed, of which the nucleotide sequences and open reading frames were proved to be correct. It showed that the recombinant proteins of Dsg4 domains EC1, EC2, EC3 and EC4 reacted to PV patients' sera, but not to normal sera.
CONCLUSION: The above data indicate that the epitopes of Dsg4 may play a role in the pathogenesis of PV.
METHODS: RNA was obtained from normal human skin tissue and then cDNA was synthesized by RT-PCR. The target gene fragments of desmoglein 4 extracellular domains (EC1-4) were amplified by PCR. With the technique of gene recombination, these target gene fragments were inserted into pET32a plasmids respectively by T4 DNA ligase, which formed the recombinant plasmids used to transform the E. coli DH5alpha competent germs. Screening of transformant germs was done by LB medium with Ampicillin. The DNA sequences of positive recombinants were then identified. The epitopes of four recombinant proteins of Dsg4 in PV patients were analyzed by ELISA.
RESULTS: Four DNA bands with all the length of 350 bp were obtained by RT-PCR. Consequently four expression plasmids of desmoglein 4 extracellular domains were constructed, of which the nucleotide sequences and open reading frames were proved to be correct. It showed that the recombinant proteins of Dsg4 domains EC1, EC2, EC3 and EC4 reacted to PV patients' sera, but not to normal sera.
CONCLUSION: The above data indicate that the epitopes of Dsg4 may play a role in the pathogenesis of PV.