Construction Of EIAV Infectious Clone With EGFP Marker

1.Background and purposeEquine infectious anemia virus (EIAV) and human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), cats immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), sheep Mehdi-Visna virus (MVV) and caprine arthritis-encephalitis virus (CAEV),belong to anti-retroviral lentivirus genus, is seriously endangering the health of human and animal pathogens.Their genome, the molecular mechanism of viral replication, antigen drift, cell macrophages, virus's life cycle, immune mechanism and the interaction between virus and host are very similar. Their common feature is against the host immune system, monocytes-macrophages or lymphocytes continued proliferation. As a member of lentivirus genu, EIAV have some unique features.EIAV incubation period only a few days to weeks, not years, and can produce high-titer viremia, severe clinical and pathological manifestations,during EIAV infection process, the new emergence of antigenic variants associated with disease recurrence, therefore, it is an excellent model to study the slow process of interaction between virus and host of antigenic variation; and a more special is chronic EIAV infection experiencing years or so into a subclinical infection, the virus-suppressed long-term infected horses have normal life, most infected horses eventually control the virus replication. Therefore, the identification of the immune protection mechanisms is important for lentiviral vaccines designe.Taking the classic 70's use of allograft passage and cell engineering method, Chinese scientists in the 20th century obtained donkey virulent DV (on horses and donkeys are 100% fatal) from a horse the super-virulent strain of EIAV LN ass body through the continuous passage of 115 on behalf of the enhanced virulence of the virus, and then acquired complete loss of virulence straight through the DV strains were cultured in vitro leukocyte domesticated donkey, while maintaining good immunogenicity and ultimately foster a successful EIAV Donkey Leukocyte attenuated vaccine DLA (Shen Rong-xian,1979), the vaccine immunized animals can not only strong and lasting immunity, and resistance to homologous and heterologous virulent attacks made virulent attacks from infected animals after the immunization. Undergo a rigorous safety inspection of widely used in China, it certificates EIAV can be effectively controlled in China. Chinese EIAV attenuated vaccine is the first time success of the slow virus diseases immunization. So far, the vaccine is the world's only successful application of slow-attenuated vaccine virus.It also makes our EIAV attenuated vaccine protection and the mechanism of attenuated lentivirus immunization becomes a hot topic in basic theoretical research. The subculture of the molecular mechanisms induced by the weak and the attenuated and virulent pathogenic interactions with animal inoculation, the immune system will enrich the basic theory of slow virus, and to include other slow viruses, including HIV vaccines provide an important theory. After the discovery of HIV-1,EIAV molecular biology research have made a lot of results, especially reverse genetics in the development of slow virus research provides a new idea.Using this technique successfully in 1998,several strains of different virulence EIAV infectious molecular clone has been built, which lay a solid foundation to reveal the virulence mechanisms of interaction with the host.EIAV receptor 1 (ELR1)is a recently discovered (2006),it belongs to tumor necrosis factor receptor superfamily proteins, till now it is the only EIAV receptor. The env gene of EIAV encodes gp90 and gp45 glycoproteins. Surface glycoprotein present in the virus membrane, size of approximately 90kDa, is a highly glycosylated protein, it constitute the virus spike in the handle, with the host cell membrane receptor interaction. The way EIAV into the target cells is:the gp90 virus membrane binding and receptor ELR1,introducing low pH conditions (pH4.8-5.3)by endocytosis into the cell.Alone could support its virulent EIAV serum and cell-adapted EIAV drug into the target cells of non-proliferation, but research shows that the reaction may require auxiliary receptor interaction, whether as receptors for HIV do exist is unconclusive.Our aim is to construct fluorescent marker into the infectious clone hopes which will help to identify potential receptors.In this paper, the existing vaccine strains of infectious clones for GFP marker, and infectious clone constructed in live rescue packaged virus particles, to study equine infectious anemia virus and host cell receptor interaction potential between The pathogenic mechanism and the reasons for vaccine protective effect. In addition, the paper part of the experiment, the fragments will be connected to SOE-PCR technique made part of the expansion and extension, through the improvement of our experiment can be completed in a single reaction tube to connect multiple segments. Hope that part of this article can bring relevant research reference for personnel laboratory.2.Research methodsSOE-PCR technique esign of overlapping primers with complementary bases in the process of realization of the two fragments amplified extension reaction, and rapid realization of synthetic genes.The technology widely in many fields of applications, include the construction of infectious clone, virus gene group sequencing the virus's genetic variation analysis, restriction fragment polymorphism analysis of genetic sub-types etc. The basis of our experimental design is also based on this.Using SOE-PCR technology, we tag a fluorescent marker EGFP to the existing infectious molecular clone of EIAV.To study the enhanced green fluorescent protein gene in living cells as a reporter gene and selection marker of the feasibility of using molecular cloning technique, the plasmid pIRES2 EGFP was cloned into EIAV infectious clone Low copy expression vector to construct the recombinant plasmid PLGgfp-3-8.GFP is a 27k Da monomer,encodes 238-amino acid,is itself a bioluminescence system, with a biological fluorescence emission after excitation to the luminescence base, the light emission process is different from other biological tissue, it needs not luciferase participation. GFP chromophore of tyrosine by serine glycine (SerTyrGly) form a ring consisting of three peptides, and only the color base coat protein in the integrity of GFP fluorescence can be issued, cut off the GFP (Even the few amino acids C terminal) can also lead to loss of light-emitting GFP capacity. GFP fluorescence excitation is independent of a specific process, does not require any cofactors, substrates or other from the jellyfish gene expression product. The Ca2+-activated light protein (Aequorin) which passed to GFP irradiates fluorescence. When GFP in prokaryotic or eukaryotic cells and are subject to Blu-ray or ultraviolet irradiation can be issued when the bright green fluorescence.EGFP is a GFP mutant with two amino acid replacement, Leu (leucine) to replace Phe64 (Phe), Thr (threonine) to replace Ser65 (Ser). The EGFP fluorescence intensity excitatied at 488nm wtGFP lamps 35 times.Eukaryotic mRNA translation generally requires 5'cap to mediate ribosome binding, except for small RNA virus family. This virus is not in its pre-mRNA cap site, but it is 600～1200bp 5'untranslated region, which contains a number of non-initiation AUG,during a long untranslated region called the internal ribosome entry site sequence (Internal ribosome entry site, IRES).IRES can recruit ribosomes on mRNA translation. The IRES and the integration of exogenous cDNA and found that IRES translation initiation independently.With a fluorescent marker in building a good infectious clone, we further carried out using 293T cells transfected rescue package to restore the virus infectious virus particles.293 cells were transfected with adenovirus E1A gene in human renal epithelial cell line,293T cells derived from 293 cells, while expression of SV40 large T antigen, SV40 replication origin and contains the promoter region of the plasmid can be replicated.Transfection kit with transfected operation can be easily transfected. High levels of protein expression after transfection by analysis of fluorescence expression can be detected more easily expressed protein. Transiently transfected 293T cells over-expression and access to intracellular proteins and extracellular (secreted or membrane) proteins and convenient way. Lentiviral expression vector contains the packaging, transfection, stable integration of genetic information needed. Lentiviral packaging plasmid can provide all of the transcription and RNA packaging into recombinant viral vectors need to leave all the auxiliary proteins. To a high titer of virus particles, need to use the same expression vector and packaging plasmids were transfected into cells, the cells for virus packaging, packing a good fake virus particles released into the extracellular medium,the centrifugation supernatant obtained After the host cells can be directly used for infection of the target gene into the host cell and following the reverse transcription, integration into the genome to a high level of expression of effector molecule. 3.ResultsSuccessful use of SOE-PCR technique was improved by adding diluted in the middle of the amount of primer, it can directly connect a 3 fragment of the existing vaccine strains of infectious molecular marker GFP cloning and sequencing results showed that a large segment of length 4.2Kb The mutation of a base number of bases. And follow-up study shows that use of the technology available into five segments of the large fragment length 8Kb connection, expanding the scope of multi-segment connection at home and abroad to conduct such a large fragment of the literature and more clips to connect not been reported, it can achieve more fragment of a large fragment of the mutation can connect the mutation rate control range.Construction of infectious clones to rescue packages in live virus particles, to study equine infectious anemia virus and host cell receptor interaction potential between the pathogenic mechanism and protective effect of the vaccine because the experiment requires further study.