Study On The Preparation Of The Anti-peptide Antibodies Against Pseudorabies Virus And The Neuronal Culture In Vitro

Pseudorabies virus(PRV) is a swine alphaherpesvirus. PRV invade and establish reactivatable infections in neurons of the peripheral nervous system(PNS) ganglia. Following transport to the cell body through axons(retrograde transport), the viral genome usually remains latent in the nucleus. Months or years later, the latent infection can be reactivated, producing newly assembled viral particles. The viral particles then travel back to the periphery(anterograde transport), where re-infection of the epithelia produces infectious virus that can spread to naive hosts. This long-distance movement of viral particles to and from the cell body engages microtubule-based fast axonal transport machinery. PRV virion invaded axons by receptor-mediated membrane fusion, which separates virion envelope, glycoproteins, and outer-tegument proteins from nucleocapsids and inner-tegument proteins. Some virion components are transported separately in axons, which influences establishment of productive or quiescent infection in the cell bodies.Ch’ng and Enquist invented triple-chamber system in order to study the directional transport of PRV between non-neuronal epithelial cells and neurons better. The triple-chamber system could separate the cell body from axon by physical barrier. Now another kind of microfluidic chamber system which different from triple-chamber system could be applied into study the effect of the protein of alphaherpesvirus on the axonal transport of alphaherpesvirus.Based on those background, The polypeptide were designed, then injected into rabbits. At last the positive serum of gB, VP16 and VP26 can be obtained. Primary cultures of hippocampus neurons were prepared from neonatal mouse and spinal dorsal root neurons were obtained from chicken embryo. We planted the DRG of chicken embryo in one compartment of microfluidic chamber system, and inoculated the recombination virus in the soma compartment where cell bodys or axonal compartment. Then the medium in the soma compartment and the axonal compartment were celebrated at certain time. The PFU of the virus in the medium were determined. Through these results the influence of PRV protein UL21 on the anterograde and retrograde transport of PRV virus particle were analyzed. The results as follow: 1. The preparation of antibody of polypeptideBased on the ammo acid sequence of gB、VP16 and VP26, we found an appropriate sequence to design polypeptide. Then the company synthetized the polypeptide based on the sequence that we designed. The polypeptide solution was injected into Newzland long-ear rabbits mixed with freund’s adjuvant. We examined the titer of serum when we were immune to the rabbits for several times. Once the titer reached the demand, the blood was obtained from carotid artery of rabbits. Then we centrifuged the blood and drew the serum. The serums were identified by western. The results proved the three serum had the corresponding antibody of different protein. 2. The primary culture of hippocampusPrimary cultures of hippocampus neurons were prepared from neonatal mouse. The hippocampus was isolated under the dissecting microscope. The cut tissues were dispersed in tissue dissociation enzymatic solution and incubated at 37℃ for 30 min. The concentration of papain is 2 mg/ml. Then we planted the single cell into six well plate or dish. The medium was refreshed every 3 days, by replacing 1/3 volume of the spent medium with an equal volume of fresh and prewarm medium. The cells were observed when cultured ten days. We identified the cells using indirect immunofluorescence assay and proved to be the hippocampus neurons. 3. The employment of the microfluidic chamber systemThe Chicken embryo spinal dorsal root neurons were planted in the microfluidic chamber system. Neuron cell bodies were cultured in the somal compartment, while axons extended through 10 mm wide microgrooves into the axonal compartment. The axons of chicken embryo spinal dorsal root neurons could extend through wide microgrooves into the axonal compartment when cultured 5 days. Then the recombinant virus which has already constructed infected the neurons in the S compartment or N compartment. The PFU of the virus in the medium harvested from soma compartment and axonal compartment at certain time were determined. The results showed that the microfluidic chamber system was a good tool to study the anterograde and retrograde of PRV.