Nghiên cứu tạo interferon ChIFN-α biểu hiện trên hệ thống tế bào nấm men Pichia pastoris và thử nghiệm trên các virus gây bệnh..

Nội dung tài liệu: Nghiên cứu tạo interferon ChIFN-α biểu hiện trên hệ thống tế bào nấm men Pichia pastoris và thử nghiệm trên các virus gây bệnh..

1. MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY SUMMARY OF DOCTORAL THESIS Specialization: Biotechnology Code: 62420201 NGUYEN THI THANH GIANG RESEARCH ON CREATING INTERFERON CHIFN-α EXPRESSING ON Pichia pastoris YEAST CELL SYSTEM AND TESTING ON CHICKEN PATHOGENIC VIRUSES Cần Thơ, 2020 i
2. THIS STUDY WAS COMPLETED AT CAN THO UNIVERSITY Scientific supervisor: Assoc. Prof. Hồ Quảng Đồ PhD. Nguyễn Đăng Quõn The dissertation was defended at the university examination committee At: , Can Tho University At hour , on date month year Referee 1: Referee 2: Referee 3: The dissertation is available at Libraries: 1. Central library of Can Tho University. 2. National library of Vietnam. ii
3. PUBLISHED PAPERS 1. Nguyen Thi Thanh Giang, Nguyen Dang Quan, Ho Quang Do, 2020. Effective in inhibiting virus causing Gumboro disease of interferon in experimental chickens. Journal of Science and Technology of Vietnam, 5(62): 48-53 2. Nguyen Thi Thanh Giang, Nguyen Dang Quan, Ho Quang Do, 2020. Establishing a procedure for determining biological activity of chicken interferon alpha (ChIFN-α). Vietnam Journal of Agricultural Science and Technology, 2 (111): 103-107. 3. Nguyen Thi Thanh Giang, Nguyen Dang Quan, Ho Quang Do, 2020. Ability to inhibit the gumboro virus in 3 weeks old chickens of chicken interferon alpha. Open University Science Journal, 15(7): 3-13. iii
4. Chapter 1 INTRODUCTION 1.1 Necessity of the dissertation In recent years, virus disease in poultry is increasingly dangerous, there are many viruses affecting the health and performance of poultry such as bird flu, Gumboro, Newcastle. Using of cytokines as support agents to enhance the effectiveness of prevention and treatment of viral diseases in poultry are concerned and increasingly feasible with the aid of genetic engineering. In which, ChIFN-α is a cytokine with great potential for application in poultry industry. ChIFN-α is part of the nonspecific immune system, which is an immunostimulating factor that is often used in low doses with vaccines to increase vaccine efficacy. The genes coding for ChIFN-α have been cloned, expressed and activated in vitro and in vivo experiment since 1994. In addition, large- volume production of ChIFN-α using the Pichia pastoris system is the best option and perfectly suited to the criteria: high efficiency; low cost; simple and convenient to use. These are also the reasons towards "Research on creating interferon chifn-α expressing on pichia pastoris yeast cell system and testing on chicken pathogenic viruses ". 1.2 The objective of the dissertation 1.2.1 The general objective of the dissertation: To create recombinant ChIFN-α, which is resistant to disease-causing viruses in chickens, aiming to be used as medicine for these diseases. At the same time, making the 1
5. procedure for determining the bioactive of recombinant ChIFN-α. 1.2.2 The specific objective of the dissertation: The study was conducted with 3 contents: (1) Creating Pichia pastoris strains had gene encoding ChIFN-α, they enable express protein on the 5L automatic fermentation system. (2) Recombinant chicken interferon alpha (rChIFN-α) was evaluated antiviral activity in vitro, in ovo and in vivo conditions. (3) Set up the procedure and determine bioactivity unit (international unit, IU/mg) of rChIFN-α 1.3 The object and limit of the study 1.3.1 The object of the study Study subjects were recombinant chicken interferon alpha protein (rChIFN-α) obtained from cultured fluids of yeast Pichia pastoris. 1.3.2 The limit of the study Recombinant chicken interferon alpha (rChIFN-α) obtained from cultured fluids was evaluated for their antiviral activity against Newcastle on UMNS / DF1 cells; evaluate the ability to prevent the replication of influenza virus in chicken embryos and evaluate the effectiveness of prevention and treatment of Gumboro disease in 3 weeks old chickens. 1.4 Time and place of study 2
6. The experiments were performed from October 2013 to August 2017 at Can Tho University, biotechnology Center Ho Chi Minh City, Institute of Vaccines and Medical Biologicals Nha Trang. 1.5 The practical scientific significance and new contributions of the dissertation Currently, viral diseases in poultry are increasingly serious. There are many viruses affect the health and productivity of poultry as bird flu, Gumboro, Newcastle. Therefore, finding a method to prevent and treat poultry diseases is an urgent requirement. In particular, the use of cytokines is receiving a lot of attention. ChIFN- is a cytokine with great potential for applications in the poultry industry. ChIFN- is part of the nonspecific immune system, has properties that inhibit the activity of mRNA leading to inhibition of viral replication. In Vietnam, there are not many studies on ChIFN- , as well as no research on ChIFN- production at pilot scale. This research provides the initial basis for the production of recombinant ChIFN- , which is resistant to viral diseases in chickens, with high potential for application in poultry farming in Vietnamese conditions, to create products for use in preventing and treating viral poultry. Therefore, the research has a high scientific and practical significance. The thesis was the first complete research showing rChIFN-α production process on fermentation systems, antiviral properties testing to some types of virus causing chicken diseases and recommendations to use prevention and treatment of virus diseases in experimental chickens. 3
7. In addition, the thesis has also given a procedure for determining the bioactivity unit (international unit, IU/mg) of rChIFN-α that it was applied regular for basic laboratory, helping research units to assess the quality substance created, promoting the process of research and production. 1.6 The layout of the dissertation The dissertation is 130 pages, including an introduction, a review of the literature, research methods, discussion results, conclusions, recommendations and appendix. The thesis has 35 tables, 60 figures và 138 references. Chapter 2 REVIEW OF THE LITERATURE The review of the literature includes 4 contents with general knowledge. Content 1 presents a profile of chicken interferon (ChIFN) including nomenclature, details of chicken interferon alpha (ChIFN-α) and application, systems used to express ChIFN-α. Content 2 presents some viruses that cause disease in chickens including Newcastle virus, Gumboro virus and Influenza A virus. Content 3 presents the protein expression system, pichia pastoris, including knowledge of pichia pastoris strains, methanol metabolic pathways and post-translation transformation ofpichia pastoris, expression vectors and mechanisms strain formation carrying many foreign copies of pichia pastoris gene. Content 4 talks about methods for determining the active unit of ChIFN-α, including presentation of virus strains corresponding to cell lines, and procedure for 4
8. determining human interferon alpha 2b titration, regulation in Vietnamese pharmacopoeia V. Chapter 3 RESEARCH METHODS 3.1 Experimental materials UMNSAH/DF1 Cell line (ATCC) E. coli DH5α strain (Nanogen Biopharma) P. pastoris SMD1168 strain (his4) (Nanogen Biopharma) Vector expression pPIC9K (Invitrogen) Virus Newcastle, Gumboro fluid (Can Tho University) Virus H5N1 fluid, free of disease embryos 10 days old (Institute of Vaccines and Medical Biologicals Nha Trang). 1 day old chicks, chick bred between Luong Phuong, Tam Hoang and industrial chicken (Vinh Long chicken hatchery). (chicken hatchery in Vinh Long) 3.2. Content and method research The dissertation had performed 3 general research contents, is presented according to the general experimental diagram in figure 3.1. 5
9. Figure 3.1 Overall experimental layout of the thesis 3.2.1 Content 1: expression of recombinant ChIFN-α on Pichia pastoris yeast This content follows the diagram in Figure 3.2. Evaluation criteria: ChIFN-α gene has 100% similar sequence in the gene bank; pichia pastoris strains carrying ChIFN-α gene grow on medium containing G418 at concentration of 10 mg/L; expression protein specifically paired with anti-ChIFN-α antibodies and when culturing by shaking flask, fermented on 5L automatic fermentation system had the target protein content of 80% of total protein. 6
10. Figure 3.2 Experimental diagram of recombinant ChIFN-α expression 3.2.2 Content 2: Evaluation of antiviral activity of ChIFN-α fluid on Pichia pastoris cells Antiviral activity of recombinant chicken interferon alpha was tested under in vitro, in ovo và in vivo conditions. (1) Resistance to Newcastle disease virus (NDV) infected on single cell layer UMNSAH / DF1 (in vitro condition). (2) Ability to inhibit influenza virus replication on 10- day-old uninfected chicken embryos (in ovo conditions). (3) Resistance to Gumboro disease virus in 3-week- old chicks (in vivo conditions). 7
11. 3.2.2.1 Antiviral ability Newcastle of rChIFN-α under in vitro condition Figure 3.3 Experimental arrangement diagram to investigate NDV antiviral activity of rChIFN-α fluid under in vitro condition Evaluation criteria: the virus causes disease in cells with specific manifestations, determining the concentration of rChIFN-α fluid that not affect to the cell survival and the rate of living cells when infecting with the treated virus group with rChIFN-α and non treatment group. 3.2.2.2 Resistance to Influenza A H5N1 virus of rChIFN-α under in ovo condition The experiment was done according to figure 3.5 Evaluation criteria: 50% Embryo Infectious Dose (EID50) of the group with rChIFN-α treatment must be lower than the group of non-rChIFN-α treated embryos (control group). 8
12. Figure 3.5 Experimental diagram to investigate the anti influenza virus activity of rChIFN-α fluid in 10-day-old chicken embryos 3.2.2.3 Resistance to Gumboro disease virus of rChIFN- α under in vivo condition The experiment is shown in figure 3.6. Evaluation criteria: rate of protected chickens, rate of survive chickens. Figure 3.6 Experimental diagram to investigate the anti Gumboro disease virus activity of rChIFN-α in 3 weeks old chickens 9
13. 3.2.3 Content 3: Establish a procedure and determine the active unit (IU/g) of rChIFN-α The procedure for determining the active unit of rChIFN-α is done according to the procedure for determining the titre of Interferon alpha 2b Vietnam Pharmacopoeia V (Ministry of Health, 2018) modified to suit laboratory conditions. Evaluation criteria: Cell concentration is suitable to bring up the well; suitable virus strains for use in experiments. Figure 3.8 Experimental diagram to design of the process for determining the active unit of rChIFN-α Determination of active unit (IU / mg) of rChIFN-α samples is arranged according to the diagram in Figure 3.9 10
14. Figure 3.9 Diagram of the treatments to determine the bioactive unit of recombinant rChIFN-α on a 96-well plate. 3.3 Data processing All raw figures are processed using Stagraphic XV.I software. Chapter 4 RESULT AND DISCUSSION 4.1 Creating a strain of P. pastoris carrying the gene encoding for ChIFN-α 4.1.1 Synthesis of genes encoding for ChIFN-α With the chicken genome pattern, the PCR reaction was performed with the forward and reverse primers to amplify 11
15. the gene segment encoding the adult peptide segment of ChIFN-α, 506bp in length. Figure 4.1 Amplifing the gene ChIFN-α by PCR reaction with primers CHIFNα-F/R. M: DNA scale; Well 1, 2: PCR product (line product at 500 bp corresponding to ChIFN-α successfully amplified from chicken genomic DNA) 4.1.2 Generation of E. coli DH5α cell lines carrying recombinant pPIC9K plasmid containing the gene encoding for ChIFN-α ChIFN-α gene segment was cut and connected to plasmid pPIC9K to create recombinant pPIC9K plasmid carrying gene, then transformed into E. coli, selected 04 colonies containing plasmid carrying gene. Figure 4.2 The restriction endonuclease reaction with EcoR I and Not I checks the presence of ChIFN-α in rpPIC9K. M: DNA scale; In wells 2, 3, 7 and 8 appear lines at position 500 bp corresponding to the size of ChIFN-α, indicating that the strain contains plasmid carrying gene. 12
16. The results of sequencing recombinant pPIC9K plasmid showed that ChIFN-α gene has completely correct sequence with theoretical sequence. 4.1.3 Creating P. pastoris cell line that carries multiple copies of the gene coding for ChIFN-α Colony screening is performed on YPD medium containing geneticin G418 (a specialized antibiotic is used for select strains of Pichia pastoris (P. pastoris) inserted with the target gene) with increased concentration gradually 2, 4, 6, 8, 10 mg/ml to screen out yeast cell lines containing multiple copies of the target gene. Table 4.1 Screening results for recombinant P. pastoris carrying multiple copies of the target gene by YPD medium containing the antibiotic G418 G418 (mg/ml) Screening medium 2 4 6 8 10 Number of colonies 112 48 19 9 5 4.1.4 Expression of rChIFN-α on shaking flask The results showed that rChIFN-α began to appear from 24 hours of methanol induction (Table 4.2). The results of electrophoresis on Tricine SDS-PAGE gel showed that the lines with size about 19 kDa corresponding to ChIFN-α were the darkest lines. Analysis by ImageJ software allows to estimate that the target protein (ChIFN- α) accounts for more than 80% of the total protein of the fermentation fluid (Figure 4.4). 13
17. Table 4.2 Total protein content generated during ChIFN-α expression in shaking flask Induction time 0 hour 24 hours 48 hours 72 hours expression (hour) Total of 3,67a 2,1 92,67b 2,0 166,67c 51,3 217c 30,1 protein (g/L) *Letter in different rows have statistically different deviations (P<0,05) Figure 4.4 The expressive protein composition of P. pastoris strains carrying the gene encoding for ChIFN-α on shake flask was analyzed by Tricine SDS-PAGE. M: protein scale; well 1-4: protein levels expressed at 0 hours, 24 hours and 72 hours. The total of protein content increased over time and reached 0.217 mg/L at 72 hours. Figure 4.5 The Western blot result of the supernatant fluid expresses ChIFN-α at the time of methanol induction. M: protein scale; at 24 hours of expression, the presence of ChIFN-α began and protein content increased with time of expression. 14
18. 4.1.5 Expression of ChIFN-α on fermentation system BioFlođ/Cellienđ 115 According to the recommendation of the supplier of Pichia pastoris strains (Invitrogen company), the induced methanol content has a great influence on the growth of yeast cells as well as the ability of protein biosynthesis. Therefore, the amount of methanol added to the medium during the induction phase of rChIFN-α expression on the automatic culture system, will be investigated at the constant injection rate of 5 ml/L (strategy 1) and the rate of injection rate increased gradually over induction time from 5ml/Lh, 7ml/Lh and 9ml/Lh (strategy 2) during the 72 ours of induction time. Detailed results of wet biomass weight (yeast cell count) and total protein content of the fermentation according to each strategy are presented in Figures 4.6 and 4.7. Figure 4.6 Wet biomass and protein content are produced over time in the fermentation using Strategy 1. 15
19. Figure 4.7 Wet biomass and protein content are produced over time in the fermentation using Strategy 2 4.2 Evaluate the activity of rChIFN-α fluid expressed on Pichia pastoris yeast cells 4.2.1 The resistance to NDV virus of rChIFN-α under in vitro condition * Effect of rChIFN-α fluid on UMNSAH / DF1 cell growth When the cells exposed to rChIFN-α at the concentrations of 12.5 àg/ml and 6.25 àg/ml, the cells had a survival rate of 97%, no difference compared with the control group (Table 4.6). However, only in the 12.5 àg/ml rChIFN-α exposure group, the cells show abnormal proliferation, cells overlap, form clusters, do not maintain normal shape cell. Therefore, rChIFN-α concentration of 6.25 àg/ml is confirmed to be safe, does not affect the life or development of cells. 16
20. Table 4.6 Effect of rChIFN-α on cell survival Dilution rChIFN-α Value OD 595 Survival rate (%) samples (μg/ml) (Mean ± SD) 1 50 0,114d ± 0,015 15,24d ± 1,97 2 25 0,242c ± 0,004 32,44c ± 0,57 4 12,5 0,723b ± 0,027 97,13b ± 3,64 8 6,25 0,725b ± 0,013 97,33b ± 1,72 16 3,12 0,732ab ± 0,018 98,29ab ± 2,45 32 1,56 0,73ab ± 0,008 98,2ab ± 1,10 Control 0 0,745a ± 0,008 100a ± 1,05 * Numbers in different rows have statistically different deviations (P<0,05).ư. Control: The cells are cultured by DMEM, 10% FBS * Effect against virus induced CPE formation by rChIFN-α Table 4.7 Effect inhibits CPE formation of rChIFN-α Value OD595 Survival rate Group (Mean ± SD) (%) rChIFN-α 1 àg/ml 0,709b ± 0,016 89,87b ± 4,70 rChIFN-α 0,1 àg/ml 0,710b ± 0,018 89,96b ± 4,65 rChIFN-α 0,01 àg/ml 0,414d ± 0,014 52,40d ± 2,59 rChIFN-α 0,001 àg/ml 0,268e ± 0,013 33,95e ± 2,43 Standard ChIFN-α 1000 0,441c ± 0,021 55,82c ± 3,03 IU/ml Control (-) 0,212f ± 0,016 26,94f ± 2,78 Cell control 0,791a ± 0,035 100a ± 0,0 * Numbers in different rows have statistically different deviations (P<0,05) Control (-): Cells were treated with NDV, not ChIFN-α Cell control: Cells were not treated with NDV, not ChIFN-α 17
21. rChIFN-α fluid has protected cells from the harmful effects of viruses, and its effectiveness is dose-dependent. The optimum concentration to protect about 90% of living cells is 0.1 àg/ml. The rChIFN-α fluid at this concentration has a better cell protection effect than the commercial products with 1000 IU/ml (Table 4.7). 4.2.2 Resistance to influenza virus of rChIFN-α under in ovo condition * Determine the total dietary dose of rChIFN-α fluid on the development of 10 day old chicken embryos Table 4.8 Effect of rChIFN-α on embryo survival Number Content Survival embryo rate Group of embryos rChIFN-α 24h 48h 72h 1 6 100 àg 6/6 6/6 6/6 2 6 10 àg 6/6 6/6 6/6 3 6 1 àg 6/6 6/6 6/6 4 3 PBS 3/3 3/3 3/3 5 3 Control 3/3 3/3 3/3 Control: Chicken embryo is normally raised, no impact The above results showed that the rChIFN-α content used from 1àg to 100àg gave 100% survival rate after 72 hours of injection. This confirmed that the concentrations of rChIFN-α fluid used in the trials did not affect embryo development * The effectiveness of rChIFN-α in prevention of Influenza A/H5N1 18
22. The results presented in Table 4.13 showed that rChIFN-α at a dose of 100g increased the resistance of chicken embryos to the experimental strain of influenza A/H5N1 virus, which showed that the amount of virus required to infect the embryos was 50% higher than 10 times. for batches of non-IFN-treated embryos (dilution 107,24 /108,24). Table 4.13 The protective effects of rChIFN-α chicken embryos when tested with influenza strain A/H5N1 Number rChIFN-α content Result Group of (EID50/0,1ml) embryos (àg/embryo/0,1ml) 1 80 100 àg 10 7.24 2 80 10 àg 10 7.54 3 80 1 àg 10 7.79 5 80 PBS 10 8.24 4.2.3 Resistance to Gumboro virus of rChIFN-α under in vivo condition * Safe dose of rChIFN-α in experimental chickens After 72 hours of droping eye/nose with rChIFN-α at the dose of 100 àg / chicken, the experimental chickens haven't showed disease, normally and no difference compared to the control group. It showed that rChIFN-α 100 àg/one is safe for experimental chickens. * The effectiveness of rChIFN-α in the treatment of Gumboro disease 19
23. The resistance of rChIFN-α is evident in the proportion of protected chickens and the proportion of live chickens. The results showed that the proportion of protected chickens in the rChIFN-α groups with the concentration of 0,1-100 àg/chicken ranged from 46,67% - 66,67%, respectively (figure 4.28). Meanwhile, in the control group (+), chickens were not protected (0%) and were significantly different from the treated groups (P=0,001). Similarly, the proportion of live chickens in groups using rChIFN-α ranged from 80 to 93,3% and higher than that in the control group (+) (60%). The two groups using rChIFN-α 100 àg/one and 10 àg/one had similar therapeutic effects. Experimental results show that using rChIFN-α at a dose of 10àg/one is most effective, limiting the morbidity and mortality rate in the treatment of Gumboro disease than other doses. Figure 4.28 The Gumboro antiviral effect of rChIFN-α across the used concentrations 20
24. * The effectiveness of rChIFN-α in the prevention and treatment of Gumboro disease The results showed that rChIFN-α (dose of 10 àg /chicken) when using for prevention, the protection rate reached 83% and when chickens were infected, the dose of 100 àg / chicken should be used for treatment, the protection rate reach 73,3%. Figure 4.31 Effectiveness of rChIFN-α when using for prevention and treatment of Gumboro disease 4.3 Procedure to determine the active unit (IU/g) of rChIFN-α 4.3.1 The cell density of UMNSAH/DF-1 is suitable for 96 well plate The cell density was investigated and insert into a 96- well plate with the goal is the cells would stick to about 80% of the cultured surface area after 24 hours. The number of cells inserted into each well was 5ì103 cells/well, 1ì104 21
25. cells/well, 2ì104 cells/well và 4ì104 cells/well, respectively. Observing the morphology at the time points after 24 hours and 48 hours put the cells on the plate and got some results (Figure 4.32; 4.33). this result showed that the most suitable amount of cells inserted into plate were 2ì104 cells/well or 2ì105 cells/ml. This result is consistent with the cell density used in the implementation procedures presented in the study of Xia (2004), Wang (2019). Figure 4.32 Cell morphology after 24 hours of culturing with amount of cells per well was 5ì103 (a); 1ì104 (b), 2ì104 (c) and 4ì104 (d). Black arrows indicate cells that are round shape, do not adhesion and die. 22
26. Figure 4.33 Cell morphology after 48 hours of culturing with amount of cells per well was 5ì103 (a); 1ì104 (b), 2ì104 (c) và 4ì104 (d). The white circle is where the cells have growth overlaps each other. 4.3.3 Determining TCID50 dose of NDV and VSV virus The VSV and NDV virus that adapted on UMNSAH/DF-1 cells were multiplied and the dose of TCID50 was determined to be used for determining unit of activity . 23
27. Table 4.20 Spreadsheet TCID50 of VSV và NDV virus fluid Parameters VSV NDV Concentration of 10-6 và 10-7 10-4 và 10-5 virus diluted CPE - above and below 50% Proportional 0,41 0,71 distance (PD) Dilute virus 10-6,41 10-4,71 concentration for CPE 50% 6.41 4.71 TCID50/0,1 ml 10 10 The significance 1 ml initial VSV 1 ml initial NDV of TCID50/0,1 virus fluid contains virus fluid contains 7.4 5.71 ml 10 TCID50 10 TCID50 The used dose in the activity determination procedure was 100 TCID50/ml, so viral samples are diluted to reach this concentration. 4.3.4 Determine the active unit (IU / mg) of rChIFN-α using NDV and VSV The active unit (IU / mg) of rChIFN-α is determined based on the modified titration procedure of Interferon alpha 2b (Vietnam Pharmacopoeia V, Ministry of Health, 2018). The tested rChIFN-α samples had protein content of 160 μg / ml, 120 μg / ml and 104 μg / ml, respectively. 24
28. Table 4.27 The calculated results of active unit and related values of rChIFN-α sample tested in 2 procedures using NDV and VSV Procedure Procedure rChIFN-α with with VSV NDV Nr 4,313 4,138 Sample 1 Ns 6,65 6,296 (160 Titres 504,939 446,541 μg/ml) Active (IU/ml) 5x106 4,5x106 Active (IU/mg) 3,1 x 107 2,8 x 107 Nr 3,832 3,945 Sample 2 Ns 6,799 6,862 (120 Titres 781,939 749,831 μg/ml) Active (IU/ml) 7,8x106 7,5x106 Active (IU/mg) 6,5x107 6,3x107 Nr 3,948 3,942 Sample 3 Ns 7,069 6,952 (104 Titres 870,158 805,36 μg/ml) Active (IU/ml) 8,7x106 8,1x106 Active (IU/mg) 8,4x107 7,7x107 If the results according to the VSV procedure are used as a reference (equivalent to 100% accuracy) for the NDV procedure, the results obtained using the NDV virus of the 25
29. samples are respectively 90,3%, 96,9%, 92,8% which corresponds to the result difference 9,7%; 3,1% và 92,8% for samples 1, 2, and 3. These results show that the difference in active units when using two viruses is within the allowable range of the health department (the tolerance titer is about 70% -150%). Therefore, it is possible to use NDV virus in the rChIFN-α active unit determination procedure. From there, the rChIFN-α active unit determination procedure will use NDV virus dose 100 TCID50, the number of cells inserted into each well was 2ì104 cells. CHAPTER 5: CONCLUSIONS AND RECOMMENDATIONS 5.1 Conclusions After doing the research, the topic has obtained a number of results: - Collected 5 yeast lines containing multiple copies of the gene ChIFN-α in the yeast genome. The yeast strain expressing rChIFN-α protein has a concentration of 1,48 g/L, of which the target protein accounts for 80% of the total protein. - When using rChIFN-α fluid at a concentration of 0,1 μg/ml, it effectively protects NDV infected cells, the number of living cells reached 89,9% compared to untreated viral control (26,9%). For chicken embryos, the dose of 100 àg/chicken rChIFN-α helps preventing infection of the Influenza A/H5N1 virus by reducing the infectious dose by 50% of the test embryo (EID50). The EID50 dose of the control group using PBS (108,24) was 10 times higher than the rChIFN-α group (107,24). For 26
30. chickens, when treating with a dose of rChIFN-α 0,1 àg/ chicken - 10 àg/chicken, the rate of protection reached 46,7 – 66,7% respectively. When using rChIFN-α fluid to prevent Gumboro disease at dose of 10 àg/chicken , the rate of protection is over 83%. The above results show that rChIFN-α is resistant to virus under in vitro, in ovo and in vitro conditions, the protective effect is base on using dose. - Process to determine the active unit rChIFN-α with specific parameters: cell density put on 96 well plate is 2x105 cells/ml (2x104 cells/well/100àl); using NDV virus at a dose of 100 TCID50/0,1ml; The active unit (IU/mg) of the same sample in repeated experiments had an error of about 10%. The active unit of rChIFN-α sample (IU/mg) is about 3,1- 8,4 107. 5.2 Recommendations - Investigating the remaining 04 strains of Pichia pastoris to select strains with high protein expression ability. - Investigating fermentation conditions on automatic fermentation systems to increase protein-collecting efficiency: investigating the composition of the culture medium, methanol addition speed. - Researching and evaluating the antiviral effect of rChIFN-α when administered intravenously or intramuscularly with mucosa to have appropriate recommendations. - Study and evaluation of rChIFN-α supplementation with virus prevention vaccines in enhancing chicken immune response and effectiveness. 27