Preparation Of Polyclonal Antibody To Yak Prion Protein Using The Expressed GST-PrP Fusion Protein As Antigen And Localization Of Yak Prion Protein In Tissue Of Bovine Encephalon

Objective: Using the prokaryotic expressed GST-BoPrP(23—242) fusion protein as the antigen to inoculate 40 BALB/c mice aged 8 weeks in clean class, so that the antiserum to Yak prion protein can be obtained, then the reactinogenicity and immunogenicity of GST-BoPrP(23—242) fusion protein will be tested; the following is to make the freezing tissue slices from 12 healthy cattle brain tissue, and determine the location of PrP in brain using the obtained antiserum as the first antigen. The result will bring the technology and the experimental materials for the determination of BSE and preparing of monoclonal antibody.Method: The DNA of Yak PrP expressed plasmid extracted from the recombinant expressed bacterium was identified by PCR amplification and two-ezyme digestion. The expressed product was obtained from the recombinant bacterium inducted under the optimized expressed condition (37℃, 1 mmol/L IPTG, 6 h), and it was determined by SDS-PAGE and Western blotting. The GST-BoPrP(23—242) fusion proteins were collected by two ways: the first is to purify and renature from the inclusion bodies; the second is to separate by SDS-PAGE. And the products were saved by freezing out in low temperature. After the collected products were mixed with Freunds adjuvant as 1:1 and emulsified, two groups clean BALB/c mice were inoculated, which were 8 weeks old, so that the antiserum was obtained. The specificity and titer of the antiserum were determined by indirect-ELISA. Using the obtained antiserum as the first antibody, and the monoclonal antibody 4C11 as the collator, the specificity of the antiserum was tested by Western blotting and indirect-ELISA with the Yak recombinant mature PrP, GST fusion protein, the extract of brain and the bacterium protein. The freezing slices of brain tissue were made, which different locations were collected from the brains of 12 catties. Then using the antiserum as the first antiboty and the monoclonal antibody 4C11 as the collator, the slices digested or undigested by protease K were dyed by ABC dyeing. The result was used to determined the location of PrP in cattle brain.Results: The determination by PCR amplication and Xho I and EcoR I digestion showed there was a expectant size line about 660 bp in gel. The Western blotting of expressed products showed there was a expectant size line on NC film, which molecular weight is about 50.2 ku, and it was responsed with 4C11. The specificity and titer of the antiserum were determined by indirect-ELISA, and the result conformed the aniserum has specificity and its titer is 1:12800. Under 400x microscope, the freezing slices dyed by ABC dyeing showed: there are some brown blots on the slices undigested by protease K, the result is positive; there are no brown blots on the slices digested by protease K, the result is negative.