Preparation,Identification And Application Of Giant Panda (SLAM/CD150) Functioning As A Canine Distemper Virus Receptor Transgenicmice

Canine distemper?CD?is an acute,high-contact disease caused by Canine distemper virus?CDV?.In recent years,the host range of CDV infection has been continuously expanding.This disease has caused the death of seven giant pandas and is considered to be the"number one killer",which brings great challenges to the protection of giant pandas.Giant pandas are an endangered wild animal in China and are not suitable for large scale animal experiments with high risk factors.It is urgent to establish a mouse disease model that can replace giant pandas for scientifics research to some extents.Signaling lymphocyte activation molecule?SLAM?,also known as CD150,is the major cell membrane receptor in the susceptible animals.In this research a transgenic mouse model expressing giant panda CD150 had been successfully established based on transgenic technology,and the genotype and phenotype of the transgenic mouse had been identified.Furthernore,we established an transgenic animal model for infection,which was inoculated the CDV isolates from giant pandas to evaluate the viral infection efficiency.The construction of a transgenic mouse expressing the giant panda CD150 molecule can provide a reliable small animal model for studying the relevant mechanisms of CDV infection of giant pandas and vaccine development.Part ?:Construction and identification of transgenic mice expressing giant panda CD150.In this study,the recombinant plasmid gpSLAM-pIRES-eGFP carrying giant panda CD150 gene was linearized and injected into mouse fertilized eggs.The fertilized eggs were then transplanted into the fallopian tubes of surrogate mother mice.After the surrogate mother gestation period is completed,the offspring's genome was extracted and the progeny mouse genotypes were identified by PCR.We has successfully obtained twelve offspring mice,including two positive micewith the positive rate?16.67%?.After hybridizing between Founder mice and wild-type mice,58 F1 mice were obtained,of which 32 were positive and the positive rate was55.17%.After genotyping the F1 mice,RT-PCR,Western blotting,and Real-time qPCR were performed to detect the expression of CD150 gene in the whole body tissue of transgenic mice.The results showed that CD150 is mainly expressed in mouse lung,heart,kidney,intestine,trachea and other tissues.We have successfully constructed transgenic mice expressing the giant panda CD150,and the giant panda CD150 gene can be stably expressed and inherited in transgenic mice.Part ?:Evaluation of CDV infection efficiency of the transgenic mice model.The F1 transgenic mice were challenged with 50?L10⁴ TCID₅₀/mL giant panda-derived canine distemper virus isolate?giantpanda/SX/2014?.The body weight of the mice was recorded every 3 days after inoculation,and whole blood,lung lavage fluid,and peritoneal washes were collected on days 7,and 21after infection for RT-PCR and Real-time qPCR.The results of RT-PCR and Real-time qPCR showed that the transgenic mice had CDV in lung lavage fluid on the 7th day after infection and the control mices did not.On the 21st day,the transgenic mice had CDV in the whole blood,lung lavage fluid and peritoneal washing fluid.The weight changes of all mice after challenge infection were not significant.The above experimentals results demonstrated that the transgenic mice carrying the transgenic giant panda CD150 increased significantly have a significant increase in the efficiency of binding to the virus,accelerating the viral infection process.In summary,this study successfully established the transgenic mice model expressing giant panda CD150 and systematically investigated evaluated the practical value of the transgenic mice,providing an alternative animal model for further research of CDV.