[Construction and identification of 2 secreted human GAD65 fragment DNA vaccines].
OBJECTIVE
To construct and identify 2 secreted human GAD65 fragment DNA vaccines.


METHODS
The GAD(190-315), GAD(490-570) cDNA and hIL-2 signal peptide cDNA were linked through overlapping PCR, respectively. The fusion gene was cloned into eukaryotic expression vector pBudCE4.1. After the DNA vaccine being determined to contain the correct target nucleotide sequence, the expression of fusion proteins was detected by Western blot.


RESULTS
The nucleotide sequence of the cloned gene was the same as the reported sequence, and their open reading fragment was correct. The products of these DNA vaccines were expressed and secreted in eukaryotic cell using Western blot.


CONCLUSIONS
The pBudCE4.1/SGAD(190-315) and pBudCE4.1/SGAD(490-570) secreted human GAD65 fragment DNA vaccines were successfully constructed, which is a foundation for immune prevention of type 1 diabetes.
Zhang
Song
S
Institute of Metabolism and Endocrinology, Diabetes Center, Second Xiangya Hospital, Central South University, Changsha 410011, China.
Zhou
Peng-cheng
PC
Huang
Gan
G
Sun
Yi
Y
Zhou
Zhi-guang
ZG
D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D005822:Genetic Vectors; D005968:Glutamate Decarboxylase / Q000145:classification / Q000235:genetics*; D006801:Humans; D016366:Open Reading Frames; D010446:Peptide Fragments / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics; D019444:Vaccines, DNA / Q000096:biosynthesis* / Q000235:genetics*
D018076:DNA, Complementary; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D019444:Vaccines, DNA; D005968:Glutamate Decarboxylase