[Secreted expression of porcine interferon beta in Pichia pastoris and its inhibition effect on the replication of Pseudorabies virus].
In order to develop recombinant porcine interferon beta with high bioactivity, the rare codes that encoded 3th, 7th and 164th amino acids of porcine interferon beta mature protein were mutant into bias codes of Pichia pastoris and then the modified gene was introduced to yeast secreted expression vector pPICZ alphaA which resulted in pPICZalphaA-PIB. The SacI linearized plasmid pPICZalphaA-PIB was transformed into Pichia pastoris X-33 by electroporation. The transformants were identified by PCR using PoIFN-beta and AOX1 specific primers. The expression of PoIFN-beta was induced with methanol. Several positive clones were obtained and the one namely B1 produced the highest level of PoIFN-beta. The B1 was further fermented in shake-flask in larger volume. The concentration of the secreted PoIFN-beta was about 60 microg/mL and its antiviral activity is about 2.5 x 10(5) U/mL, so the specific activity of porcine interferon beta produced by the Pichia pastoris is approximately 4.17 x 10(6) U/mg. The expressed supernatant was concentrated and identified by SDS-PAGE and Western blot. There are two major proteins with respective molecular mass of approximately 25 kDa and 28 kDa in the supernatant. The results of Western blot indicated that the two proteins were positively reacted and manifested well PoIFN-beta antigenicity. In contrast with the deduced theoretical molecular mass value of PoIFN-beta, the expressed two major proteins were larger which maybe due to the difference of glycosylation. The antiviral effect of recombinant porcine interferon beta (rPoIFN-beta) on Pseudorabies virus (PrV) was studied in the present experiment. The result indicated that rPoIFN-beta could effectively inhibit the replication of PrV in MDBK cells, especially during the early phage of the virus replication.
Cao
Rui-Bing
RB
Key Laboratory of Animal Disease Diagnostic and Immunology, Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, China. crb@njau.edu.cn
Zhou
Guo-Dong
GD
Zhou
Hai-Xia
HX
Bao
Jing-Jing
JJ
Chen
Pu-Yan
PY
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D015870:Gene Expression; D005818:Genetic Engineering; D011558:Herpesvirus 1, Suid / Q000187:drug effects* / Q000502:physiology*; D016899:Interferon-beta / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology*; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D013552:Swine; D014779:Virus Replication / Q000187:drug effects*
D016899:Interferon-beta