The Development Of Rabies Genetic Engineering Vaccine And The Reform Of Gene Therapy Vector

Rabies is an acute, lethal, zoonotic infectious disease which is caused by rabies virus(RABV). Once infected, there is mostly 100% mortality rate. At present, there is no effective treatment,except that prevaccination is the best way to prevent this disease. As a single and negative strand RNA virus, rabies virus’ s genome mainly encods 5 kinds of structural protein which, respectively, are nucleoprotein(N), phosphoprotein(P), matrixprotein(M), glycoprotein(G), RNA polymerase(L) from 3’end to 5’end. Among these glycoprotein is not only the only viral protein which can induce the body produce virus neutralizing antibody(VNA) that are protective against rabies virus, but also decide virus cellular tropism and the ability the virus adsorption, so that the rabies virus have strong neurotropism.Neurodegenerative diseases are caused by loss of neurons or their myelin, and they are more and more deteriorated with the time going, to cause dysfunction, which cause serious damage to the elderly health. Due to the difficulty across the blood-brain barrier(BBB), drugs for the diseases are extremely limited. More and more scientists are keen to explore ways, like gene therapy, to deal with these diseases. But, at present, the vectors used to transfer and express some genes such as lenti-virus, retrovirus, adeno-associated virus(AAV) etc, are weaker to target nerve cells. 1. The development of rabies subunit vaccineGiven the characteristics of the G protein can induce the body to produce neutralizing antibody, this project try to research a new type of subunit vaccine which adeno-associated viral vector with the adavantages of high safety, low immunogenicity, long duration of expressing foreign gene, etc, expresses the G protein. The G proteins were from SADL16 vaccine strain G protein(including AA194, AA333 were mutated(mt) and not mutated type(wt)), B2 C strain G protein and N2 C strain G protein. At the same time, to improve the quantity of G protein expression, woodchuck hepatitis virus after transcriptional regulatory elements(WPRE) was inserted into the expression vector. The r AAV as vaccines which can express G protein were packaging on 293 T cells, and after titering, G protein expression test, they were inject into 6-week old female Balb/c mice by intramuscular immunization, then respectively at the end of 1th, 2th, 3th, 9th month detect the neutralizing antibody level of mice. Results showed that immuned mice to produce neutralizing antibodies, and compared with the control group, there is an obvious advantage. To investigate if the vaccine can protect mice from rabies infecting, the mice were challenged with 50LD50 of CVS-24 and observed daily for 20 days. Poison attack experiment showed that the vaccine could provide effective protection against rabies for the body. Compared with other vaccines, the vaccines have the advantages of low cost, simple immune program(a single immune), storage, transportation conditions require low(4℃), long duration of antibody levels, etc. 2. The development of G polyclonal antibodyBecause of being difficult, in vitro, to express and purify the G protein, rabies virus G protein polyclonal antibody(Pc Ab) can be rapidly prepared by this method. Above all, results also showed that with this method G protein Pc Ab was fast prepared. 3.The reform of rabiesvirus as a gene therapy vectorBased on the rabies virus has stronger neurotropism, the rabies virus was tried to gradually transformand improve the safety and finally as carrier of gene therapy for neurodegenerative disease. First, the brain derived neurophic factor(BDNF) and nerve growth factor(NGF) were knocked into rabies virus genome which the G protein had been deleted. To improve the safety of the vector, the rabies virus M protein or M and P protein were knocked out. The rabies with G protein being deleted can be rescued on the B7 GG cell. However, because there is not the cell line can be used to rescue virus whose M, G protein or M, P, G protein were deleted, the cell line with expressing M, P, G protein need to build based on B7 GG cell line by CRISPR technology.