Cloning And Sequence Analysis Of Complement Component C3d Between Domestic Animals And Fowl & Research On Immune Adjuvant Of The Recombinant Chicken C3d

In the production practice, the traditional vaccine (attenuated vaccine, inactivated vaccine) play an important role on animal disease prevention and control. But in recent years, the immunization of such vaccines exposed immunity effect ineffective and other defects, the development of more secure, efficient and cheap novel vaccine become very urgent and necessary. Genetic immunization or DNA vaccination has initiated a new era of vaccine research. The technology involves the inoculation of plasmid DNA into a living host to elicit an immune response to a protein encoded on the plasmid. The potential advantages of DNA vaccines include the induction of cellular and humoral immune responses, flexible genetic design, lack of infection risk, stability of reagents, and the relatively low cost of production in amicrobial host. However the gene vaccine could not provocate high titer and long-lasting immune response. Several approaches were explored to enhance the immunity of DNA vaccines such as using molecular adjuvant. The molecular adjuvants mainly are interferon, interleukin, thymosin and C3d, etc., which C3d as a novel molecular adjuvant is of growing concern.Complement C3 is the core factor of the classical pathway, alternative pathway and lectin pathway. Cleavages of C3 have extensive immune function. C3d is the final cleavage product of C3. It's been thought to be the least cleavage which can't be digested by protease and can covalent link to the antigen. It can attach to the CR2 which is on the surface of antigen-presenting cells (APC), so it cuts down the activation threshold of lymphocyte and elevates the immunity of the antigen. C3d has been defined to be an effective molecular adjuvant.Based on the above mentioned research background, the present study investigated the relevance and difference between different poultry and domestic animals in gene level; to study the adjuvant effect of recombined C3d protein, the immune efficacy of the FCA-E.coli antigen or the combined inoculation of oil-adjuvant inactivated vaccine and recombined C3d protein against NDV were observed; investigated the immunogenicity and protective efficacy of DNA vaccine encoding the F gene fused to complement P29 (complement C3d receptor binding domain), and evaluated the immunogenicity to the fusion protein of F with different copies of chicken homolog complement C3d; and performed an control experiment upon the adjuvant effect between pcDNA3.1-F-ChC3d-p29.6 and recombined C3d protein. The main research content is as follows:1,Cloning and Sequence Analysis of Complement Component C3d between Different Domestic Animals and Fowl. Total cell RNA was extracted from the liver tissue of domestic animals and fowl, and the cDNA of C3d was amplified by reverse transcription polymerase chain reaction (RT-PCR). The cDNA fragments were directly cloned into pMD18-T plasmid, and the recombinant plasmids pMD18-T-C3d were identified by restriction end nucleases digestion and sequencing. The cloned C3d genes and CR2 binding region on C3d were compared between the mammalia and poultry. The results of sequence analysis indicated that the homology of complement C3d gene between domestic animals and fowl is big, and the phylogenetic tree showed C3d had varieties in species, more close relationship, more close evolution. Structural analysis in CR2 binding region indicated that there were 29 amino acids in fowl, which had low homology to mammalians; this indicated that the CR2 binding region had genus-specificity. The homologous amino acids were less than 40% between fowl and mammalians, however there were more homologous among mammalians, nearly 80%. In a word, our research proved that the complement C3d and the CR2 binding region had the genus-specificity. The deduced amino acid sequence and the structure of C3d were analyzed using biological software. By the software such as homogenous model of ESyPred 3D, the molecular characteristics and the three-dimensional structure of C3d's tertiary structures also anticipated in this study. It will lay a foundation for further studying on construction of gene vaccines using chicken C3d as molecular adjuvant.2,Construction,prokaryotic expression and purification of chicken's complement C3d. In order to study C3d's function of enhancing immunity, the cDNA of Roman chicken C3d was amplified by reverse transcription polymerase chain reaction (RT-PCR). The PCR products were cloned into plasmid pET-32a. After confirming its rightness by PCR and analysis of restriction endnucleases, the recombinant plasmid was transformed into E. coli BL21(DE3) strain. The culture was induced by 1.0 mmol/L IPTG for several hours and analyzed with SDS-PAGE. The result shows that C3d gene was expressed successfully in E. coli and the fusion protein existed in supernatant had immunogenicity. The expression products were purified by (NH4)2SO4 fractionation and Sephadex G-200 gel filtration. This report constituted a solid foundation for further researches of chicken C3d.3,Verificated the immunized reinforcement of recombined chicken's complement C3d. Glutaraldehyde was used as cross linking agent to combine the recombined C3d with the antigen of E. coli. The complex was used to immunize 11 days aged SPF chickens, and the control group chickens were injected with the FCA-E.coli antigen. The blood of chickens was drawn at 2,3,4,5,6,7,8,9 weeks after they were immunized respectively. Using ELISA to determine the content of antibody in the serum. The total complement activity of the serum was determined at the same time. It was demonstrated that during the first three weeks IgG titer induced by FCA (FCA-E.coli)was higher than those induced by C3(C3d-E.coli), but it decreased rapidly after reaching a peak(OD=0.273±0.040) at the end of 4th week since they were immunized,and it decreased to 0.200±0.050 at the end of 9th week. But the antibody level of the chickens immunized with C3d was increasing gradually all the time,from 0.099±0.030 (determined at second week) to 0.275±0.020 (determined at 9th week). Thus the adjuvant effect of C3d was different from that of FCA, it could induce the production of memory cells and make the antigens stimulate immune cells consistently and stably. This study provided the theoretical basis for developing effective bacterial vaccines for the poultry.4,Protection of chickens from Newcastle Disease by recombined C3d protein combined with oil-adjuvant inactivated vaccine. To study the adjuvant effect of recombined C3d protein, the immune efficacy of the combined inoculation of oil-adjuvant inactivated vaccine and recombined C3d protein against NDV were observed. The results showed that: not only the proliferation response of the T lymphocytes from thymus and the B lymphocytes from bursa in chickens immunized with combined inoculation were significant stronger than those of chickens immunized only with the oil-adjuvant inactivated vaccine during all experiment period, but also the titers of HI antibody of chickens inoculated with both the oil-adjuvant inactivated vaccine and recombined C3d protein were significant higher than that of chickens inoculated only with the oil-adjuvant inactivated vaccine. When challenged with field strain at 49 days post-inoculation, the chickens immunized with combined inoculation obtained 100% protection, but chickens immunized only with the oil-adjuvant inactivated vaccine only obtained 83.3% protection. It suggested that the combination of trivalent oil-adjuvant inactivated vaccine and DNA vaccine could highly enhance the immune response and increase the immunity against NDV.5,Construction and expression of DNA vaccines containing F gene against Newcastle Disease Virus with C3d-P29 as molecular adjuvant. C3d is the final cleavage product of C3 and fusion to C3d enhances the immunogenicity of the antigen. CR2/CD21 plays a very important role in its function. P29 is the gene encoding the binding area to CR2 of C3d.After cloning the C3d cDNA of Roman layer used the liver as the source of mRNA; a pair of primers was designed to subclone the P29 gene to the pUC19 plasmid. Several tandems of P29 were constructed in the pUC19 plasmid used a pair of isoschizomers-Bam H I and Bgl II. Digested the pUC-P29.n to get the gene of P29.n, and then cloned the products to pCDNA3.1 (+) plasmid. After this, the F gene of Newcastle disease virus (NDV) was cloned through RT-PCR and inserted to the upstream of the P29.n which is in the pCDNA-P29.n, and the DNA Vaccines containing F gene against NDV with C3d-P29 as molecular adjuvant were constructed. The expression of pcDNA-F-C3d-P29.n was detected by immunofluorecent assay (IFA). This research, which was the further study complement C3d as the molecular adjuvant, had laid the foundation in developing and using chicken C3d.6,The research about the recombined chicken complement C3d-P29 specially enhanced the complement's total titer and the content of complement C3. For confirmation the function of chicken complement C3d as molecular adjuvant, this experimental compared the difference in the complement level in the chicken blood serum and the complement C3 content among the blank comparison and the recombined plasmid which includes the heterogeneous animal (for example mouse) the CR2 region, to test whether the recombined chicken complement C3d-P29 could specially enhanced the complement's total titer and the content of complement C3. Divides into five groups according to the construction eukaryon expression recombined plasmid to carry on the immunization injection differently to the chicken, there were PBS group,pcDNA3.1 group,pcDNA3.1-F group,pcDNA3.1-F-ChC3d-P29.6 group and pcDNA3.1-F-MC3d-P28.6 group. The complement's total titer and the content of complement C3 were determine. Results showed that the content of complement C3 of different recombined plasmid were: PBS group 0.34±0.005mg/mL, pcDNA3.1 group 1.47±0.008mg/mL, pcDNA3.1-F 1.70±0.005mg/mL, pcDNA3.1-F-MC3d-P28.6 group 1.78±0.007mg/mL, pcDNA3.1-F-ChC3d-P29.6 group 4.18±0.008mg/mL respectively. The content of complement C3 of pcDNA3.1-F-ChC3d-P29.6 group was lower than the other groups obviously. The discrepancy was extremely significant(p≤0.01). And the recombined chicken complement C3d-P29 could specially enhanced the complement's total titer and the content of complement C3, The discrepancy was extremely significant(p≤0.01).7,Study on immunological responses to eukaryotic recombinant expression vector plasmid with C3d as molecular adjuvant on chickens. The present study investigated the immunogenicity and protective efficacy of DNA vaccine encoding the F gene fused to complement P29 (complement C3d receptor binding domain), and evaluated the immunogenicity to the fusion protein of F with different copies of chicken homolog complement C3d. The DNA Vaccines containing F gene against NDV with C3d-P29 as molecular adjuvant were constructed in the formerly research (pcDNA3.1-F-C3d-P29.2,pcDNA3.1-F-C3d-P29.4,pcDNA3.1-F-C3d-P29.6 and pcDNA3.1-F), and its safety and potency were evaluated. The SPF chickens were immunized with NDV DNA Vaccines and inactivated NDV vaccine respectively. To evaluate vaccine's immune efficacy, using lymphocyte proliferation test (MTT) to measure lymphocytes proliferation in vitro, using HI to measure serum special IgG and using ELISA tests to detect IgG titers. At last, In order to investigate the immnoadjuvant effect of pcDNA3.1-F-ChC3d-p29.6, rChC3d was co-administrated with inactivated NDV vaccines. The results show that DNA Vaccines which containing NDV F gene and C3d-P29 as molecular adjuvant were safe, it could induce humoral and cell-mediated immune response and mucosal immunity strongly. The results of the potency tests conformed that the vaccine could produce good protective effect. And the results of the dynamic changes of T lymphocyte transformation were determined by MTT method showed that the C3d-P29 adjuvant could significantly promote T lymphocyte transformation, especially pcDNA3.1-F-C3d-P29.6, which was similar to oil adjuvant at the most time points and was stronger at some time point. Proliferation responses between pcDNA3.1-F-ChC3d-p29.6 and rChC3d of chicken Periphery blood lymphocytes were measured by MTT assay. Peak values of the dynamic changes to T lymphocyte transformation in the rChC3d co-inoculated group were attained at 4th week after initial inoculation, which was earlier than the group inoculated with pcDNA3.1-F-ChC3d-p29.6, but could not went on as long as the pcDNA3.1-F-ChC3d-p29.6 group. These results presented that pcDNA3.1-F-ChC3d-p29.6 could be employed much more effective immunoadjuvant to conventional vaccines against NDV compare with rChC3d. To date, published research into the adjuvant activities of C3d has been limited to experiments in mice, using antigens unrelated to diseases occurring naturally in these species and there is little information about chicken C3d. This research will lay a foundation for further studying on investigation of the chicken C3d.