Luận án Nghiên cứu biểu hiện kháng nguyên hemagglutinin (HA) tái tổ hợp của virus cúm A / H5N1 và đánh giá tính sinh miễn dịch trên gà

Biểu hiện thành công gen ha1, ha1-2 của virus cúm A/H5N1 dưới dạng dung hợp với gen trx có vị trí cắt của enterokinase và thrombin (trx-te-ha1, trx-te-ha1- 2) trong E. coli BL21. Ở quy mô bình tam giác, trong môi trường LB, nhiệt độ 30oC, nồng độ chất cảm ứng IPTG 0,5 mM. Protein TrxHA1E được tổng hợp chủ yếu ở dạng thể vùi, hàm lượng protein tái tổ hợp đạt 126 mg/l. Hiệu giá HI trung bình huyết thanh gà gây miễn dịch bằng TrxHA1E đạt 2,2-2,8 log2. 2. Biểu hiện thành công gen ha1, ha1-2 dung hợp với gen trx trong nấm men P. pastoris dưới dạng có (trx-te-ha1, trx-te-ha1-2) và không có vị trí cắt của enterokinase và thrombin (trxha1, trxha1-2). Hiệu suất biểu hiện gen trxha1, trxha1-2 cao hơn so với trx-te-ha1, trx-te-ha1-2. Khả năng sinh đáp ứng miễn dịch của các protein tái tổ hợp Trx-TE-HA1, TrxHA1, TrxHA1-2 tương đương nhau. Điều kiện thích hợp cho quá trình biểu hiện gen trxha1: môi trường BMMGY, pH 5-6, nồng độ methanol cảm ứng 1%. Hàm lượng TrxHA1 dịch lên men biểu hiện quy mô bình tam giác và trong nồi lên men 10 lít đạt 14 mg/L và 84 mg/L.

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main of avian influenza H5N1 virus in E. coli BL21 in the fusion form with thioredoxin. 2) Expressing genes encoding HA1 globular and HA1-2 domain from avian influenza virus H5N1 in P. pastoris. 3) Selection suitable parameters for high HA expression 4) Evaluating the immunogenicity of recombinant HA protein in chicken trials. Results: The fusion protein of TrxHA1E and TrxHAl-2E with the molecular mass of 57 and 66 kDa were synthesized successfully in E. coli BL 21 cells in LB medium at 30°C under the regulation of T7 promoter which was induced by IPTG. The 108 fusion proteins were synthesized in inclusion body at 30 o C under induction of 0.5 mM IPTG. The effects of temperature (22, 25, 30 and 37 o C) and concentration of induction factor IPTG (0.1, 0.5, 1.0, 1.5 and 2.0 mM) were investigated. TrxHA1E was expressed at 30 o C accounts for 20% of total proteins, expression levels of TrxHA1E approximately 126 mg/l by ELISA. The solubility of TrxHA1-2E was over 70% as expressing at the temperature 25 o C and yield of TrxHA1-2E was 86 mg/l. Recombinant TrxHA1E protein then was separated from host soluble proteins by sonication combined with centrifugation, then solubilized in 2M urea had antigenicity to bind with antibody against HA of virus H5N1. TrxHA1E had capable of triggering the production of neutralizing antibodies in chicken when they were immunized with recombinant protein by subcutaneous injection or intranasal immunization. Hemagglutination inhibition (HI) titers of chickens sera after 2 weeks of booster immunization were 2.2-2.8 log2. The results showed that TrxHA1E had immunogenicity although the HI titers were at low levels. In P. pastoris, the fusion proteins were expressed in the two form: (1) containing protease sites thrombin (T) and enterokinase (E) that located on the linking sequence between trx and ha1 (Trx-TE-HA1), trx and ha1-2 (Trx-TE-HA1- 2); (2) removing the two protease sites on the linking sequence (TrxHA1, TrxHA1- 2). Both of Trx-TE-HA1 and Trx-TE-HA1-2 were expressed at low level and proteolyzed in the cultivation medium. In order to further improve the expression level of HA recombinant protein from A/H5N1 avian virus in P. pastoris we optimized the HA1 protein coding sequence and expressed it in P. pastoris X33, but foreign protein expression was also not stable. By gene construct removing the protease sites on the linking sequence reduced the proteolysis of the fusion protein and increased the expression proficiency in comparision with the prevous construct. In order to further improve the expression level of recombinant HA protein from A/H5N1 avian virus we optimized the HA5.1 protein coding sequence and expressed it in P. pastoris X33. After optimization, Codon Adaptation Index (CAI) value was improved from 0.69 to 0.98, without modifying the amino acid sequence 109 of the encoded protein. The synthetic mha1 with a length of 1 kb was inserted in to pPICZαmha1 then expressed in P. pastoris X33. MHA1 recombinant protein of 50 - 70 kDa was synthesized at 30 o C under induction of 1% methanol during 72 hours. Recombinant HA protein had biological function to agglutinate chicken’s blood cells at and had antigenicity to bind with antibody against HA of H5N1 virus. The structures of recombinant HA protein by removing the two protease sites on the linking sequences were also expressed in P. pastoris SMD1168. The recombinant proteins were produced with higher productivity and more stable. Different induction media including BMM, BMMY, BMMG, BMMGY and different induction ingredients were used to get higher fermentation yield of TrxHA1. Western blotting of fermentation culture supernatants with differ-ent media of the recombinant P. pastoris strain with anti - HA antibody revealed an obvious difference in the amount of the TrxHA1 protein produced between the different media, with a greater apparent yield being obtained from the suspension culture in modified BMMGY media with productivity estimated to be 3.4-fold higher than in standard media BMM and BMMY. Cultures in shake flasks provided expression levels of TrxHA1 approximately 14 mg/l. The recombinant protein was suffered from glycosylation and protease processing. The fed-batch fermentation at 10 L scale was developed for high-yield secretory expression of the recombinant Hemagglutinin protein. The TrxHA1 concentration of the clarified broth was found to be 84 mg/L. The TrxHA1 was partly purified by precipitation with 20 and 60% ammonium sulfate (mass/volumn) or purified using Co-NTA affinity chromatography. The doses of 100 μg TrxHA1 were immunized for two week-old chickens by subcutaneous injection or by intranasal administration for assessment of TrxHA1 immunogenicity. In subcutaneous injection chickens groups, haemagglutinin inhibition (HI) titer of chickens sera harvested after 2 weeks of booster immunization reached 7.0-7.2 log2. In intranasal immunization groups, HI titers were about 6.6-7.0 log2. 110 In conclusion, the study has demonstrated that P. pastoris can be used to express influenza TrxHA1 and TrxHA1-2 proteins with antigenicity and immunogenicity obtained. The expressed proteins were successfully purified from the expression culture without additional extraction steps, suggesting the feasibility of a cost-effective and large-scale production for vaccine purpose. Chickens vaccinated with TrxHA1 protein produced high neutralizing antibody titers, at rates comparable with the licensed inactivated H5N1 vaccine. Therefore, this system may act as an attractive and effective candidate vaccine to produce protein for avian influenza vaccines. 111 NHỮNG CÔNG TRÌNH CÔNG BỐ LIÊN QUAN ĐẾN LUẬN ÁN 1. Nguyễn Thị Thảo, Văn Thị Như Ngọc, Lê Thị Thu Hồng, Võ Viết Cường, Đỗ Thị Huyền, Trương Nam Hải (2010) Tối ưu hoá biểu hiện HA5.1 của virus cúm A/H5N1 trong nấm men P. pastoris SMD 1168. Hội nghị khoa học 35 năm thành lập Viện Khoa học & Công nghệ Việt Nam. Nxb. Khoa học tự nhiên và Công nghệ, 238-243. 2. Võ Viết Cường, Trần Thị Nhài, Đỗ Thị Huyền, Trương Văn Dung, Trương Nam Hải (2013) Nghiên cứu tinh chế và đánh giá khả năng sinh đáp ứng miễn dịch của protein HA5.1 từ virus cúm A/H5N1 biểu hiện trong Escherichia coli. Tạp chí Công nghệ sinh học, Số 3 (13):319-325. 3. 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