| Bovine viral diarrhea(BVD)is an important infectious disease caused by bovine viral diarrhea virus(BVDV).It mainly infects animals such as cows,deer,sheep and pigs.The disease is widely distributed and has caused huge economic losses to the livestock industry in worldwide.Presently,experienced countries in the West mainly use inactivated and attenuated vaccines to prevent the disease.However,Chinese research on BVDV started late,and vaccine development is relatively fall behind.Only one inactivated vaccine is approved for clinical use.Moreover,its immunogenicity is poor,and the cost of immunization is high.Therefore,there is an urgent demand to develop new vaccines with high immunogenicity and low cost.This provides a new method for the prevention and control of BVDV in China.BVDV is a single-stranded RNA virus with a size of about 12.3kb.Its open reading frame encodes a long polyprotein that is cleaved into 12 mature polypeptides by viruses and host proteases.The NS5 B protein is located at the carboxy terminus of the polyprotein.It has RNA-dependent RNA polymerase activity.It is essential for its genome replication.Without the NS5 B protein,the BVDV genome cannot replicate.BVDV cannot proliferate in the host cell,so it loses the ability to continuously infect.Therefore,this feature can be used to construct a BVDV vaccine strain lacking NS5 B.This will provide new ideas for the development of Chinese BVDV vaccines.Constructing a full-length infectious cDNA clone is a necessary way to obtain NS5 B gene deletion vaccine.Although there have been reports before,all rescue methods for BVDV require in vitro transcription,in vitro synthesis,and RNA transfection.The process is cumbersome and RNA is easily degraded,which requires high operation technology.Therefore,it is necessary to develop a simple and easy-to-operate reverse genetic system to rescue BVDV.In order to solve the above problems,in this paper,we carried out the following research:(1)In order to detect BVDV virus,we constructed a prokaryotic expression vector of NS5 B protein.IPTG inducers were used to induce protein expression.The purified protein is used as the main immunogen to inoculate mice.Mice will undergo an immune response,producing polyclonal antibodies.The study found that NS5 B protein is mainly expressed in the form of inclusion bodies in E.coli.And the truncated NS5 B has higher expression than untruncated.This polyclonal antibody was used to detect BVDV and showed good specificity.(2)Construct NS5B lentiviral expression vector,and then use electroporation transfection method to transfer it into BHK cells.Puromycin was used to screen positive monoclonal cells expressing NS5 B.And gradually expand to BHK cell line stably expressing NS5 B protein.(3)In order to facilitate the detection of the immune effect of the BVDV vaccine,we constructed a mouse model of BVDV infection.Wild-type BVDV was used to infect mice by oral method.The expression of BVDV in the spleen was detected by Western Blot and immunofluorescence.HE staining was used to observe the pathological damage of BVDV to its tissues.The study found that oral vaccination can infect mice with BVDV.Moreover,the expression of BVDV was the highest on the fifth day after infection.Interestingly,BVDV infection had no obvious pathological damage to liver,heart and spleen tissues of mice.(4)This article simplifies the BVDV reverse genetic operating system.The basic operation is to add a hammerhead ribozyme sequence before the 5′UTR of its genome,so that the RNA transcribed from the host cell can accurately cut off the excess sequence at the 5′ end.Adding the hepatitis D virus ribozyme sequence after the3′UTR can ensure the integrity of the 3′ end of BVDV.After being improved,it can directly transfect cells to produce BVDV without in vitro transcription.This makes BVDV rescue better than previously reported systems.Subsequently,the constructed BVDV infectious RNA expression vector was transfected into BHK cells to obtain its infectious RNA,that is,rescue BVDV.After MDBK cells were infected with BVDV,they were blindly transmitted for 5 generations.By observing the cytopathic condition and PCR test to determine whether the rescue BVDV is positive.Furthermore,the infectivity of rescue BVDV was analyzed by immunofluorescence and Western Blot.The study found that rescue BVDV was positive and infectious.However,there was no obvious cytopathic condition after cell infection.And the virus titer is lower.After infecting the mice,they were tested by Western Blot.We found that rescue BVDV can infect mice.(5)The NS5B gene is deleted based on the BVDV whole genome clone,thereby constructing a BVDV infectious RNA expression vector lacking NS5 B.In this paper,the rescue and analysis of BVDV were further studied,and the idea of constructing NS5B-deficient vaccine was proposed.This provides a new method for rescue of BVDV and lays the foundation for the development of BVDV-deficient vaccine strains.This will have a great significance to our national defense control BVDV. |
