| Buffalo(Bubalus bubalus, Bb), Mammalia Bovidae Buffalo species, is suitable for paddy cultivation. Buffalo milk contains higher protein, amino acids, butterfat, vitamins, trace elements than in Holstein milk with nutrition value of it is almost twice of Holstein milk. As a class of high nutritious drink, it is gradually becoming the "new favorite" of human consumption. It has potentially of great commercial value. Buffalo meat, especially young buffalo meat, is characterized by fresh, delicious and low fat content. In addition, Buffalo horn can be used to make traditional Chinese medicines. Buffalo resources are abundant in China, according to FAO statistics, by the end of 2003, the amount of buffalo is 22.759 millions in China, has risen to the third in the world.Tuberculosis is a zoonoses caused by Mycobacterium tuberculosis. Human may infect with TB from humans by contacting buffalo with TB, or drinking unpasteurized buffalo milk with TB. Buffalo TB has been the potential threat of the development of milking buffalo. So far, buffalo quarantine of TB by the use of tuberculin skin test(TST) which is widely used in dairy cattle is never calibrated. Therefore, development of a new method for the dection of buffalo tuberculosis is in urgent need.Interferon is a class of proteins which have anti-virus, anti-tumor properties and immune regulation effect. IFN-γis interferon typeⅡ, IFN-y release in vitro to detect of TB is one of the most promising detection methods. It is a method for IFN-γqualitative and quantitative analysis based on the specific anti-IFN-y monoclonal antibody. Foreign scholars have applied antigen-specific IFN-y test to the detection of a variety of animal diseases. Relatively speaking, studies t of antigen-specific IFN-γtest of specific diseases for buffalo are rare.In this study, we cloned, expressed, purified and analyzed IFN-γgene of buffalo(BbIFN-γ), prepared monoclonal antibody and polyclonal antibody of BbIFN-γ, established double-antibody sandwich ELISA method for BbIFN-γ.1. Cloning and expression of the BbIFN-γin Escherichia coli and YeastTotal RNA was isolated from buffalo peripheral blood lymphocytes, which were stimulated with Con A. Basing on sequence of Guangxi swamp type of bufflo IFN-y mRNA published in GenBank, a pair of primers were designed, cDNA was amplified and cloned into the prokaryotic expression pET-30a vector. Then after being transformed it into E. coli BL21 and induced by IPTG, soluble protein with the size of about 22kD was expressed. According to sequencing results, the codons were changed based on Pichia pastoris codon usage bias and synthesized by the company, subcloned it into yeast shuttle plasmid pPICZaA, transformed it into yeast competent cell GS115, induced by methanol to express protein with the size of about 18kD.2. Determination of anti-VSV and IBRV activity for BbIFN-γIn order to identify whether the expressed of prokaryotic and yeast BbIFN-γhas antiviral activity or not, the ability to inhibit the replication of VSV or IBRV in MDBK cells was detected. Prokaryotic and yeast expression of two proteins anti-VSV activity was proved to be 2.05×105U/mL and 7.44×105U/mL, anti-IBRV virus activity was proved to be 5.22×104U/mL and 2.61×105U/mL. Although the two recombinant BbIFN-γhave anti-viral activity, the BbIFN-y from yeast yielded higher anti-IBRV activity than the prokaryotic protein.3. Preparation and identification of BbIFN-γmonoclonal antibodyIn this study, we mixed the two proteins and immunized Balb/C mice. The proteins were independently used to screen hybridoma cells able to produce monoclonal antibody to BbIFN-γ. Finally five monoclonal antibodies with high titer, named 1C3,2C3,3E7, 4G6,4H4 were obtained. The titers of ascites were 2.56×104,5.12×104,1.28×104, 2.56×104,5.12×104 respectively. Western blot analysis showed that five monoclonal antibodies can bind BbIFN-γspecifically. Indirect ELISA showed that McAbs have no reaction with other antigens, which further confirmed the specificity is good.In addition, we mixed the two proteins and immunized rabbits, and rabbits hyperimmune sera were obtained. After purifying, polyclonal antibody IgG can be obtained. Western-blot analysis showed that polyclonal antibodies can bind BbIFN-γspecifically.4. Establishment of double-antibody sandwich ELISABy using McAb 2C3, PcAb and Goat anti Rabbit IgG-HRP, a double-antibody sandwich ELISA was established. The sensitivity of this method to detect BbIFN-γreached 125pg/mL, The establishment of this method laid the foundation of the development of the BbIFN-γdetection kit and diagnosis of buffalo TB or other diseases by using BbIFN-γdetection kit. |
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