Screening Of Nucleic Acid Aptamers Against Rabies Virus L Protein And Its Preliminary Application

Rabies is a zoonotic infectious disease caused by rabies virus infection.The mortality rate is closely 100%.There is no effective treatment for rabies has been developed by far.Rabies virus belongs to the genus rabies virus of the Rhabdoviridae family,is a single-stranded negative-strand RNA virus.Rabies RNA mainly encodes five structural proteins: nuclear protein(N),phosphoprotein(P),matrix protein(M),glycoprotein(G),and large transcriptase protein(L).L protein of the rabies virus is an RNA-dependent RNA polymerase,which is essential for the production of all viral proteins,including the L protein itself.The L protein is involved in the transcription of viral RNA by affecting its multiple enzyme activities,including transcriptase activity,capping and polyadenylation enzyme activity.L protein mediates viral genomic RNA replication through its replicase activity.That's why L protein as a potential candidate for molecular targets.Aptamer is an oligonucleotide with specific affinity for the target molecule screened by SELEX technology.It has become a research hotspot in the clinical treatment and diagnosis.Therefore,screening the aptamers for rabies virus L protein has important potential significance in clinical diagnosis and treatment of rabies.In order to express and purify the recombinant L protein of rabies virus.Firstly,the rabies virus L protein gene sequence was retrieved from NCBI using Primer5 software,and specific primers with Eco R I and Xho I restriction sites were designed.The amplified target gene was inserted into the p ET32 a vector and verified by PCR and Sanger sequencing,and an expression vector was successfully constructed.The recombinant rabies virus L protein was induced by IPTG and detected by SDS-PAGE.The results showed that a large amount of recombinant protein was expressed.After purification and renaturation,a large amount of recombinant rabies virus L protein was obtained.The results of Western blot experiments showed that the molecular weight was 42 KD.In addition,mice were immunized with recombinant L protein to prepare polyclonal antibodies,result shown that polyclonal antibodies reactivity with virus-infected cells well,which further indirectly proved that the recombinant L protein sequence expressed was correct.Furthermore,the aptamer was screened using the recombinant rabies virus L protein,and the recombinant rabies virus L protein was immobilized on a nickel affinity column,and the screening was performed based on the SELEX technology.After 10 rounds of screening,the library was ligated into p MD19 T vector,transformed into competent Escherichia coli cells,and Sanger sequencing is performed for each single clone.After analysis of the sequencing results,50 sequences were obtained,and 4 sequences that repeat many times were obtained.The structure of the aptamers was predicted by Mfold software.The results showed that there were stem-loop structures in the aptamer,and Apta-RABV-L-1,Apta-RABV-L-10,and Apta-RABV-L-33 have similar structures.The secondary structure of Apta-RABV-L-49 is quite different from those of other nucleic acid aptamers.Finally,ELONA technology was used to characterize the specificity and affinity of the four aptamers.The results showed that BSA,ZIKV-Pr M and m CCL5 proteins were not cross react with aptamers.And because ZIKV-Pr M and m CCL5 both contain a histidine tag,it shows that the nucleic acid aptamer does not cross react with the histidine tag.The aptamers Apta-RABV-L-33 and Apta-RABV-L-49 can specifically recognize the recombinant rabies virus L protein.The dissociation constants KD between Apta-RABV-L-33,Apta-RABV-L-49 and the target molecule were both nano-level,indicating that these two aptamers had higher affinity for the recombinant rabies virus L protein.At the same time,the indirect immunofluorescence method was used to detect the expression of N protein in the HEP-Flury stains infected N2 a cells treated with nucleic acid aptamers,indicating that the above-mentioned aptamers inhibit proliferation of rabies virus.In this study,recombinant rabies virus L protein was successfully prepared,and the recombinant rabies virus was used as a target molecule to screen two nucleic acid aptamers capable of specific high affinity with the target molecule using the SELEX technology.The aptamer exhibits high affinity to rabies virus L protein,it inhibits proliferation of HEP-Flury strains.Aptamers has advantages of cheapness,low immunogenicity,and easy medication compared with tranditionnal antibodies,which may lay the foundation for targeted treatment of rabies.