Cloning,Expression And Activity Identification Of Pokeweed Antiviral Protein PAP-I Gene In Wuzhi Mountain Of Hainan Island

Pokeweed Antiviral Proteins(PAPs)belongs to the type I Ribosome Inactivating protein,which has RNA N-glycosidase activity,can exclusively cut a adenine from 28 s rRNA specific site A4324 of eukaryotes 60 s ribosomal subunits which make it difficult to combine with the extension of protein synthesis factor EF-2,thus preventing the protein synthesis in cells.Recently many researchers are paying much attention on the molecular structure,protein chemistry and antiviral mechanism etc of PAPs for their natural broad spectrum antiviral protein which could resistance to a variety of plant and animal viruses.That could provide theoretical and API support for the development of antiviral drugs.At present,the PAPs which used for anti-human hepatitis B virus infection research has made great progress and has shown its application prospects in the field of medicine.PAPs has significant effects on the prevention and treatment of viral infection on plants and animals,but the premise is need to get a lot of PAP antiviral protein with highpurity and high activity.The PAPs is expressed in different tissues and developmental stages which could not meet the needs of mass production.If the gene can be cloned,constructed to the expression vector and expressed in escherichia coli,and established efficient denaturation,renaturation technology,innovation and the establishment of a rapid antiviral biological activity identification method with high sensitivity and high specificity,expression and screening of high-purity,high biological activity of recombinant antiviralprotein PAP which can get rid of the limit of time and space,and better meet the needs of research and industry.The pokeweed from Wuzhi Mountain of Hainan was used as experiment material in gene cloning and protein purification:1 n The total RNA was extracted from the fresh spring leaf,use RT-PCR technique to obtain the cDNA sequence,then design primers with enzyme digestion sites to clone the PAP-I gene,the result showed that there were no base mutations and frameshift mutations which was verified by sequencing.2.Bioinformatics analysis of PAP-I gene showed that the full-length cDNA coding region of PAP-I gene was 942 bp,encoding a 313 amino acid protein,N-terminal signal peptide consists of 22 amino acids,C-terminal composed by a stable region which consists of 29 amino acids.The gene has a typical PAP family conserved domains,belongs to the ribosome inactivating protein.Subcellular localization prediction result of PAP-I gene is positioned in extracellular.According to PAP-I protein physicochemical properties speculation,the PAP-I protein formula is C1577H2493N415O469S14,its relative molecular weight is 35.22 kD,an isoelectric point(pI)is 8.86.Theoretical derivation of its half-life is about 30 h,the unstable parameter is 34.39,a stable protein.Hydrophilic average:-0.189,which predicted that the protein is hydrophilic proteins,and the fat index is 90.29;The secondary structure of PAP-I protein was mainly random coil,followed by the extension of the chain structure and a-helix,?-comer shares the minimum proportion;The homology modeling three-dimensional structure of PAP-I protein through SWISS-MODEL software to obtain the predicted three-dimensional structure of amino acid sequence;The results of amino acid sequence of PAP-I analysis showed that PAP-I protein has a transmembrane domain;PAP-I belongs to hydrophilic protein through analysis of hydrophilic and hydrophobic speculation;The potential phosphorylation sites analysis of PAP-I protein shows that PAP-I protein can be phosphorylated by a serine kinase,threonine kinases and tyrosine kinases phosphorylation,in order to achieve the regulation of its functions.3.Constructed the prokaryotic expression vector pET30a-PAP-I which was using the gene of Wuzhi Mountain pokeweed.4.The expression and purification methods of pET30a-PAP-I in E.coli BL21(DE3)showed that 0.6 mmol/L IPTG is the optimum induction concentration and the optimal induction time is 4 h.5.Through the wheat germ extract transcription/translation coupling system,according to the antiviral mechanism of pokeweed antiviral protein and the characteristics of inhibition of protein synthesis,the new identification of biologically active recombinant protein technology was established through the mechanism of the luciferase enzyme inhibition.This method is not only with higher sensitivity and specificity,but also faster and easier compared with the traditional method of identifying the biological antiviral activity.6.The purified natural protein was used as an antigen,the complete adjuvant and incomplete adjuvant was used interchangeably to immunize rabbits in this test.And the high titer polyclonal antibodies were obtained at last which overcomes the difficulty of PAP-I protein with low immunogenicity and provides an efficient and specific antibodies for the identification during the expression and purification of PAP.7.The target protein was purified by Ni-Agarose affinity chromatography.SDS-PAGE electrophoresis analysis showed that the purified protein was a single band,which proved the purification effect is good.The results showed that the protein is PAP-I by SDS-PAGE and Western verification.8.The renaturation of expression protein was using the method of column on circulation renaturation.The activity of the recombinant protein PAP-I was proved through wheat germ extract transcription/translation coupling system.The results showed that the inhibition rate to the expression of luciferase was 84.1%when the concentration of the recombinant protein was 0.2 ?g,and while the concentration of the recombinant protein was 1.2 ?g,the inhibition rate could reach up to 99.9%which showed a strong inhibition of protein synthesis activity.In summary,the system of the prokaryotic expression of PAP-I proteins,the purification technology,denaturation and renaturation key technology of efficient recombinant PAP,quantitative detection and biologically active detection technology of recombinant proteins PAP were established in the present study which provide technical and material support for the development of antiviral drugs.