Construction and application of a yeast expression system for thymosin alpha1.
We want to construct a yeast expression system for thymosin alpha1 (Talpha1) to make the orally administered Talpha1 preparation possible. The whole Talpha1 DNA fragment was obtained by PCR. After being digested with restriction enzymes, it was cloned into pYES2 vector. Sequencing was performed to identify the recombinant. The sequence of Talpha1 in recombinant coincided with the original one reported in Genbank. When pYES2-Talpha1 plasmid was transformed into yeast, galactose instead of glucose was used to induce Talpha1 expression. Western blot was performed to identify the quality of the expressed Talpha1. Dried yeast containing pYEST2-Talpha1 was fed to Balb/c mice whose immunities were inhibited by cyclophosphamide in advance. Synthesized Talpha1 peptide was used as positive control and empty yeast was used as negative control. Compared with the negative control group, both dried yeast containing pYEST2-Talpha1 and synthesized Talpha1 peptide can significantly increase the CD8+ level (22.74 +/- 1.09 and 18.77 +/- 4.72 vs 7.49 +/- 2.14, p < 0.01), while both of them had little effect on the CD4+ lymphocytes (61.86 +/- 6.94 and 65.91 +/- 4.78 vs 57.93 +/- 10.40,p > 0.05). We concluded that a high effective yeast expression system for Talpha1 was constructed successfully and the Talpha1 protein expressed by this system can improve CD8+ level in immune inhibited mice.
Chen
Feng
F
Institute of Infectious Diseases, 1st Affiliated Hospital, Medical College of Zhejiang University, Key Lab. of Infectious Diseases, Ministry of Public Health, Hangzhou, 310003 China. xinlanchen222@hotmail.com
Chen
Xiang-Ming
XM
Chen
Zhi
Z
Jiang
Han-Liang
HL
Pan
Xiao-Ping
XP
Hu
Zhong-Rong
ZR
Liu
Rong-Hua
RH
Chen
Xiao-Ming
XM
D000818:Animals; D015153:Blotting, Western; D016516:CD4-CD8 Ratio; D018414:CD8-Positive T-Lymphocytes / Q000187:drug effects; D002999:Clone Cells / Q000187:drug effects; D003001:Cloning, Molecular; D003520:Cyclophosphamide / Q000633:toxicity; D005434:Flow Cytometry; D005612:Freeze Drying; D015870:Gene Expression; D005822:Genetic Vectors; D007166:Immunosuppressive Agents / Q000633:toxicity; D007274:Injections, Intraperitoneal; D051379:Mice; D008807:Mice, Inbred BALB C; D016133:Polymerase Chain Reaction; D011897:Random Allocation; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D013010:Sonication; D013601:T-Lymphocytes / Q000187:drug effects; D000077596:Thymalfasin; D013947:Thymosin / Q000031:analogs & derivatives* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D007166:Immunosuppressive Agents; D011994:Recombinant Proteins; D013947:Thymosin; D003520:Cyclophosphamide; D000077596:Thymalfasin