Cloning Of TLR2,TLR7 Genes And Expression,Preparation Of Polyclonal Antibody And Tissue Expression Analysis Of TLR7 Protein In Chinese Alligator

Due to destruction of habitat and environmental factors,the number of Chinese alligator population decline rapidly.Today,after the establishment of Chinese Alligator Nature Reserve in Xuancheng,Anhui;Tangshan,Nanjing;Changxing,Zhejiang and other places,being protected by artificial breeding,the Chinese alligator population has rapidly expanded to millions,and the farming experience and techniques are also be more mature.Although Chinese alligator is rare and the animal under first-class state protection,but so far,compared to a variety of bacterial and viral diseases of crocodiles reported abroad,reports on the Chinese alligator diseases and infection mechanisms of our country are still few.Therefore,to carry out research on the Chinese Alligator immune disease-related proteins(such as Toll-like receptors)will contribute to understanding their innate immune functions and mechanisms,thereby reducing disease and promoting health farming of Chinese alligator.At the same time,using modern molecular biology knowledge and techniques to analysis and develop scientific management strategies,is also an important part of the current Chinese alligator protection.In this article,TLR2 and TLR7 conserved regions of different species were being compared,and pairs of degenerate primers were designed.In order to amplify target fragment from cDNA in the liver of Chinese alligator in Tangshan,Nanjing farm by RT-PCR,and to do homology analysis with the corresponding nucleotide the predicted amino acid sequence of other species,and to construct phylogenetic trees.Amplification results showed,TLR2 and TLR7 fragments were 526 bp and 433 bp,respectively.Sequencing results showed that,compared with the corresponding TLR2,TLR7 sequence of the latest data of Chinese alligator in Changxing,Zhejiang,the identity both reached up to 99%at nucleotide level.Homology analysis showed that the Chinese alligator,the Alligator mississippiensis,turtles and birds are in high homology;Phylogenetic analysis showed that Chinese alligator and Alligator mississippiensis are on the same branch,so there is a close genetic relationship between the two.The TLR7 gene of Chinese alligator was cloned into pET-3 2a(+)to construct recombinant plasmid pET-32a(+)-TLR7.The recombinant plasmid was transformed into E.coli BL21(DE3),and the bacterium was induced with IPTG at 37 ℃.It was demonstrated by SDS-PAGE that the protein was expressed in E.coli BL21(DE3).Then the expressed protein was purified by cutting protein containing slices from SDS-PAGE gel and immunized in New Zealand rabbits to prepare polyclonal antibody against TLR7.SDS-PAGE showed that the recombinant bacteria could express at approximately 35ku.Detecting the antibody titer by indirect enzyme-linked immunosorbent assay(ELISA),the results showed that rabbit anti-TLR7 recombinant protein serum titer was about 1:25600.Western-blot results suggested that the recombinant protein was expressed correctly,and can react with specific antibodies,which has good immunogenicity.To extract total protein from liver,spleen,lung,kidney and small intestine tissues in hibernation and non-hibernation,after SDS-PAGE and transferred to PVDF membranes,we regard prepared rabbit polyclonal antibody as the primary antibody,β-actin as internal control,to detect the relative expression level of TLR7 protein in different tissues with Western-blot,while extracting total RNA from liver,spleen,lung,kidney,small intestine and muscle,to detect TLR7 mRNA expression level differences with real-time quantitative PCR assay.The results showed TLR7 expression was tissue-specific,and expression level of mRNA and protein were not exactly the same,but the expression levels in hibernation are generally lower than the non-hibernation.