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The Development and Antiviral Activity of Recombinant Bovine Interferon Alpha and Gamma

Author: LiXueRen
Tutor: ChenZuoYan
School: Nanjing Agricultural College
Course: Preventive Veterinary Medicine
Keywords: Bovine interferon gene cloning prokaryotic pichia pastoris antiviral activity
CLC: S859.79
Type: Master's thesis
Year: 2005
Downloads: 191
Quote: 1
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Abstract


At present, there are many infectious diseases that cumber the thriving and the updating of the cow-farming industry. So there are urgent wishes and needs to develop the new generation of the safe and truly effective antiviral drugs and immune potentiator or adjuvant to enhance the heath of cows and improve the efficacy of the current vaccine. Interferon are cytokines that function as antiviral factors and immune regulators, which are naturally produced by the immune system of mammal immediately following viral infection or viral vaccination. The interferon system is the first frontier of defense against viral infection in mammals. Interferon are classified into two types, namely Type I and Type II interferon, according to the diversity of amino acid sequence, the antigenicity and the type of cells from which they are produced. Interferon alpha and interferon beta are the major members of type I interferon and the only one of Type II interferon is interferon gamma. Type I interferon is famous as the potent antiviral factors, while interferon gamma were manifested as the stronger regulators for immune and antiviral potent factors with only a litter lower potency than Type I interferon. The recombinant human interferon had been successfully used to cure the viral diseases and tumor since decades ago. Recent years, the bovine interferon’s antiviral function and the adjuvant potency for vaccine have been found and conformed through many experiments and trials. One aim of this experiment is developing the recombinant bovine interferon alpha, gamma with gene and protein engineering technique, of which we have the fully independent intellectual property right. Another aim of the present study is proving their antiviral potency, in order to pave the way for the production of the engineering bovine interferon in the large scale and their application in cow farming industry.This study includes:1. With the reverse transcription polymerase chain reaction (RT-PCR) the DNA sequence encoding the cattle’s interferon-gamma (BovIFN-γ) and the signal peptide was amplified, from the total RNA of the lymphocytes stimulated with concanavalin A in the peripheral blood of bovine, which was then cloned into vector pMD18-T and sequenced. The sequencing result showed that there is 100% homology among the documented sequences and sequence reported here, which was successfully inserted into the expressing plasmid PBV and was highly expressed in E coli. The SDS-PAGE result showed that the cloned recombinant protein was expressed in the form of inclusion bodies in the E.coli cell with molecular weight of 16kDa and was amount to 32% of the whole protein in the E.coli cell, which was subsequently dissolved in 7mol/L guanidine chloride and renatured with dilution in refolding buffer containing 0.5mol/L guanidine chloride. In order to obtain pure protein, the renatured boIFN-γ was desalting by Hiprep 26/10 and purified by Hiprep Sephacryl S-200 chromatography. The purified product could inhibit the cytopathic effect, that verified it have the high cytokine activation.2. Total RNA was isolated from bovine peripheral blood lymphocytes, which were stimulated with New Castle Disease Virus. Then the bovine IFN-α cDNA was amplified by reverse transcription polymerase chain reaction. The amplified fragment was cloned into vector pMD18-T and then sequenced. The result indicated that the cloned gene was a mature bovine BoIFN-a gene, which had the identities of 98% with the BoIFN-a1 gene published in the Genebank. The cloned gene fragment was inserted into Pichia Pas tor is expression vector pPICZaA. The recombinant plasmid was linearized with Sac I and then transformed into X-33 strain by electroporation. Whether the BoIFN-a gene was integrated into the alcohol oxidase promoter (AOX1) locus on yeast chromosome was verified by amplification with AOXl primers. The SDS-PAGE result showed that cloned recombinant protein was expressed in the supernatant with molecular weight of approximated 23kDa. The expressed protein was larger, which was maybe due to the differ

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Veterinary Pharmacology > Veterinary drugs
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