| Recently,With the reasons of the intensive and high-density rearing of poultry, a great numbers of global diseases have been broken out,such as ALV-J、MDV、REV and so on.ALV-J, characterized by medulloblastoma and other cell malignancy, is a sort of Infectious neoplastic disease. Since ALV-J was found in China, with the growth of infection host ranges and the advance of onset ages, the types of tumor increase sharply, that has brought serious economic losses in poultry. Currently, there is no effective means of prevention and treatment of ALV-J, so it is necessary for seeking a kind of drugs to inhibit ALV-J viral effectively.Avian β- defensin(AvBD) is a class of host defense proteins that widely present in avian. The main structure features of AvBD is six cysteine residues that form three pairs of disulfide bonds. AvBD is the first defense of microbial invasion in body. With broad-spectrum anti-bacterial, anti-viral and anti-fungal biological effects, AvBD plays an important role in the innate immune system in poultry.To further study the distribution in tissues and anti-virus effect of the chicken AvBD,we use PCR methods, amplified AvBD6 genes from chicken liver tissue. The sequencing result was to be amino acid sequence homology analysis with avian β- defensins and birdβ-defensins, drawing molecular phylogenetic tree. The results showed that AvBD6 gene precursor peptide by is 67 amino acid residues, and the cDNA is 204 bp, comprising six cysteine stable, located in 1-5,2-4,3-6 and linked to form 3 molecular disulfide bonds,inverted form a stable parallel 3 shares β- sheet structure C-X4-8-C-X3-5-C-X9-13-C- X4-7-CC,The highest amino acids homology is AvBD7 up to 62.7%.Chicken AvBD6 was subcloned into the expression vector pGEX-6p-1, and the recombinant plasmids were transformed into Rosseta(ER2566) competent cells, induced by IPTG. Expression products of chicken AvBD6 fusion protein is present asinclusion bodies.Recombinant protein inclusion bodies of chicken AvBD6 were purified, and then were immunized in New Zealand white rabbits for preparation polyclonal antibodies. Thepolyclonal antibodies were measured by ELISA The polyclonal antibody titer was up to 1:211. To detect the distribution and location of chicken AvBD6, immunohistochemistry(IHC) was conducted in various tissues in different age of SPF chickens. Chicken AvBD6 was present in myeloid cells of bone marrow, breathing capillary epithelial cells of lung,cytoplasm of hepatocytes of liver, epithelial cells of renal tubular, intestinal villus epithelial cells of the duodenum, red pulp of the spleen. Real-time quantitative PCR method was established to detect chicken AvBD6 m RNA. Dynamic expression pattern of AvBD6 in different day-old SPF chickens was detected. We found that: ①The expression Levels of AvBD6 in various tissues and organs was detected. The expressions of AvBD6 in the bone marrow, lung, kidney, liver, spleen tissues and organs are higher than those in other tissues and organs. ②With the growth of age, the expression of AvBD6 presents "wave" shape regularity. ③ AvBD6 is expressed in many tissues and organs, especially in the bone marrow, which may play an important protective role for the bone marrow hematopoietic system.In order to define whether AvBD6 has the function of anti-ALV-J viral or not, dialysis protein refolding method has been used for obtaining a natural structure of AvBD6 recombinant protein. The study shows the AvBD6 recombinant protein has a good effect on inhibiting ALV-J viral, the inhibition efficiency is up to 95.6%.In this study the immunohistochemical methods, Real-time PCR have been used for testing the distribution of AvBD6, the expression of various organizations respectively. A preliminary study of inhibiting the ALV-J viral infection by AvBD6 recombinant protein in vitro can lay the foundation of the study of the biological function of AvBD and the innate immune system of avian. |
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