| As a new type of viral nucleic acid receptor, LSm14 A plays an important role in innate immunity as an important antiviral immune molecule,which is important to resisting the invasion of many kinds of pathogens. The present there is little research about LSm14 A and Newcastle disease virus, and The mechanisms underlying innate antiviral responses with unknown.This study had amplificated different sources of chickens LSm14 A gene and detailed analysis and functional prediction from the gene sequence, by constructing of expression plasmids of chicken LSm14 A, using real-time fluorescence quantitative PCR to illustrates different tissues between distribution and expression characteristics of LSm14 A from the molecular level, and then research Green shell layers LSm14 A and different type virus of NDV, preliminary studied clearly the effect of NDV on the transcription level of chicken LSm14 A. The results lay the foundation between the relationship of to deeply study NDV and the host innate immune system, deepen people’s understanding of regulation of virus host key signaling molecules, to provide new ideas for the prevention and treatment of NDV, and has important significance for the exploration of the innate immune system.1. Gene Cloning and Bioinformatic Analysis of the LSm14 A from ChickensThe study by RT-PCR obtained c LSm14 A gene fragment from Frizzle chicken, Green Shell Layers and Gaojiao chicken, resulting a band was consistent with the expected size is about 1386 bp. The PCR amplificated from 3 kinds source c LSm14 A gene fragment cloning and sequencing, expected results and comments are 1386 bp, encoding 461 amino acids and a termination codon. The sequencing results by NCBI blast analysis discovery and sequence similarity of Gallus（Gen Bank accession number: NM001012778.1） were 100%, 99%, 99%.To compare and analysis the obtained 3 kinds of chicken source amino acid sequences and reference gallus series, the similarity of 4 kinds of chicken source amino acid were very high,didn’t find amino acid deletion and insertion, alignmented the amino acid sequences with Gallus source and then discoved: there is only a small number of the amino acid had mutated,and Green Shell Layers amino acid sequence of the 78 aa, 135 aa, 180 aa, 300 aa lead the mutations C/R, G/R, Q/H, E/K, Gaojiao chicken the 116 aa lead the mutations C/R. Construction of evolutionary tree on c LSm14 A gene sequence from 3 kinds of chicken source with c LSm14 A gene sequence from other animal species and analysis discovered that 3 kinds of chicken source sequence with Gallus in the same branch,with common mallard, penguin,golden eagle, killdeer aves animal shave have closer genetic relationship; with monkey,mouse, pig and Silurana LSm14 A relationship is distant. The LSm14 A amino acid comparison of different species showed that the N-terminal of LSm14 A was highly conserved. 3 kinds of chickens source c LSm14 A protein had not transmembrane, containing Sm domain during 1-75 aa, containing FDF domain during 289-399 aa. 3 kinds of chickens source c LSm14 A protein had not signal peptide, both were non-secretory proteins. Obtained in the study about the LSm14 A gene level, protein structure, protein secondary structure and subcellular localization of relevant information, It’s provided theoretical foundation for further study on the gene prokaryotic expression and tissue expression.2. Prokaryotic Expression and Immunogenicity Analysis of the LSm14 A gene from ChickensAccording to the Frizzle chicken c Lsm14 A gene sequence, gene was cloned into the prokaryotic expression plasmid p Cold I, and the recombinant plasmid were transformed into BL21 competent cells,induced and expressed by IPTG, the recombinant protein was purifird by the His-tag Ni-NTA spin columns, the recombinant protein was purified r His-c LSm14 A parts immunized mouse and rabbits of corresponding polyclonal antibody, using ELISA and Western blotting to analysis the immunogenicity of c LSm14 A. The SDS-PAGE assay showed the the recombinant protein distrbuted mainly in inclusion body: The titers of serum mouse-anti-c LSm14 A and rabbit-anti-c LSm14 A antibodies were both higher than 1:3200.The results of Western blotting showed that the recombinant protein displayed an immune responsed by the His-antibody, mouse-anti-c LSm14 A hyperimmune serum and rabbit-anti-c LSm14 A hyperimmune serum. The band weighted about 19.6k Da in the western blotting was matched with the result from SDS-PAGE. A large number of Rabbit anti-c LSm14 A and Mouse anti-c LSm14 A polyclonal antibody were successfully prepared,the results to further analyze the expression of c LSm14 A and to provide the reserve of c LSm14 A antiviral effect.3. Construction of c LSm14 A Eukaryotic Expression vectors from Chickens and in vitro expressionIn this study, based on the acquired c LSm14 A sequence from the Frizzle chicken, the target fragment was cloned to the eukaryotic expression vector pc DNA3.1（+）. Subsequently,the eukaryotic expression recombinant pc DNA3.1（+）-c LSm14 A was obtained.The chosen Lipofecter lipidosome was used to transfected HEK293 cells.According to the results,c LSm14 A from the chicken could express in the HEK293 cells, mainly distributed within the cytoplasm.4. Study on Tissue Distribution from chickens c LSm14 A and the expression level of NDV influences on LSm14AThe study aims at detecting the distribution of different tissues of c LSm14 A in Green Shell Layers with q PCR to verify the distribution of different tissues of molecules of c LSm14 A. The result shows that c LSm14 A has different levels on transcription expressions among tissues of livers, thymuses, kidneys, brains, small intestines, lungs, spleens, hearts and bursa of fabricius of Green Shell Layers and these tissues have significant differences on transcriptional level. Among them, the transcriptional level exsiting in bursa of Fabricius and thymuses is higher, and the transcriptional level of bursa of fabricius is the highest; he transcriptional levels of kidneys, brains, small intestines, lungs, spleens and hearts are relatively low, thirdly, the level in livers is the lowest.To take the measure of EID50 in chosen viral strains before they are infected is to infect unimmunized checkens by utilizing different sources and virulences of NDV F48E8, La Sota,P3, H2 and TN strain. To utilize the way of q PCR is to detect the change of the transcriptional level of c LSm14 A in different time and tissues by collecting diffreent tissues of 12 h, 24 h, 48 h and 72 h to extract tissue head RNA and compoun c DNA with inverse transcription. The result turns to be that the transcriptional level of the checken’s c LSm14 A gene m RNA rises rapidly after it is inoculated NDV virus, the transcriptional level is higher than before in general, and the molecule of the checken-orgin c LSm14 A is induce by NDV.The result can lay the foundation of deeply studying the innate immunity relationship between NDV and its host, to make people know more about virus regulates and controls its host’s key signal molecule, and to provide a new idea prevent NDV.Conclusion:1. The study successfully cloned gene c LSm14 A from the Frizzle chicken, Green Shell Layers and Gaojiao chicken. By sequence analysis that discovered the gene was highly conservative, and compared the NCBI Gallus reference sequence similarity was highly to99.6%-100%. The secondary structure prediction of 3 kinds of chickens source c LSm14 A gene encoding protein, containing a large number of alpha helices and beta angle. Biological software analysis founded that 3 kinds of chickens source c LSm14 A protein without transmembrane structure, containing Sm and FDF motifs, are non secretory protein.2. The study using The original expression system successfully expressed Frizzle chicken protein c LSm14 A, obtained higher titer polyclonal antibody. Prokaryotic expression protein polyclonal antibody can specifically recognize purification, and it had good immunogenicity.3. This study had successfully built the eukaryotic expression recombinant pc DNA3.1（+）-c LSm14 A originated from the Frizzle chicken, and demonstrated that the c LSm14 A originated from the chicken could express within the HEK293 cells and mainly distributed within the cytoplasm.4. This study successfully proved the c LSm14 A in chicken liver, thymus, kidney, brain,small intestine, lung, spleen, heart, bursa of fabricius tissue transcription level characteristics.The results of experiment show that c LSm14 A in chicken body special central immune organ’s capsule in the highest transcription level. The rapid increase of c LSm14 A transcription level in the early stage of NDV infection showed that chicken c LSm14 A had important roles in early antiviral infection. |
