Recombinant expression and immunological characterization of Streptococcus iniae antigens
- Abstract
- 현재 국내 양식 어류 특히 우리나라 양식 넙치에 발생하는 연쇄구균증은 Streptococcus iniae, Streptococcus parauberis 및 Lactococcus garvieae등 3종의 연쇄구균종에 의해 발생한다. 이러한 연쇄구균증은 심각한 폐사를 일으키고 있는데, 이에 대한 뚜렷한 예방 및 치료대책이 없는 실정이다. 연쇄구균증에 의한 질병과 폐사를 예방하고자 이들에 대한 백신 개발이 필요한데, 본 연구에서는 단위항원을 이용한 subunit vaccine을 개발하기 위해 S. iniae로 부터 GAPDH, α-enolase, Streptolysin S, iron uptake ABC transporter등의 항원 후보 유전자를 선정하여, 재조합 단백질 발현 및 분자 면역학적 특성을 분석하였다. 분석결과로부터 S. iniae의 GAPDH와 α-enolase는 cell wall protein 및 plasmin(ogen) binding protein이라는 것을 알 수 있었다. hemolysin or cytolysin 작용을 나타내는 Streptolysin S (SagA) 및 세균의 생존 및 독성에 중요한 역할을 하는 철의 수송에 관련된 piaA에 대한 항체는 S. iniae에 대한 유의적인 bactericidal activity를 나타내었다. 이러한 분석결과로부터 본 연구에서 선정한 4개의 유전자는 추후 연쇄구균에 대한 subunit vaccine개발에 있어서 주요한 방어항원으로 활용될 수 있을 것으로 여겨진다. 본 연구에서는 또한 위 4개의 연쇄구균 유전자를 항원으로 하여 어류에 효율적으로 delivery 하기 위하여 Edwardsiellar tarda ghost bacteria의 표면에 이 항원들을 표면에 발현시키는 vaccine system을 제작함으로써 앞으로 어류 연쇄구균증에 대한 백신개발에 있어서 항원성 및 효율적인 전달을 가능케 하였다.
During the last decade, sporadic and epidemic outbreaks of fish diseases due to gram-positive cocci have been reported world-widely, and are responsible for significant economic losses in the fish farm industry. In Korea, warm-water streptococcosis have been a main cause of mortality in cultured fish, and 3 species - S. iniae, S. parauberis, and L. garviae - have been identified as the causing.
To design potent and universally applicable subunit protein vaccines, it is necessary to identify those antigens that are recognized as nonself by the immune systems of a wide fish population or species during infection. In the present study, 4 genes - glyceraldehyde-3-phosphate dehydrogenase (GAPDH), α-enolase, streptolysin, and ABC iron transporter - were selected as subunit vaccine candidates. The full open reading frames (ORF) of the selected 4 genes of S. iniae were cloned and produced as recombinant proteins to characterize their function and to obtain antisera. In the investigation of vaccine efficacy, the organisms immunized with recombinant proteins showed higher serum bactericidal activity than those of control.
As a nonliving delivery system with the capacity to be loaded with foreign antigens, bacterial ghosts are a safe alternative to live-bacterial delivery system. The display of heterologous antigens on the surfaces of pathogenic bacteria is of considerable value for the development of combination vaccines, and would be advantageous for the induction of antigen specific antibody responses when using attenuated or inactivated recombinant bacteria for immunization.
In this study, to generate combination vaccine system, we have generated E. coli and E. tarda ghosts displaying the above selected antigens of S. iniae on the outer membrane, and expression of S. iniae antigens on the outer membrane was confirmed by immunoblot.
- Issued Date
- 2007
- Awarded Date
- 2007. 2
- Type
- Dissertation
- Publisher
- 부경대학교 대학원
- Alternative Author(s)
- Kim, Min-Sun
- Affiliation
- 부경대학교 대학원
- Department
- 대학원 어병학과
- Advisor
- 김기홍
- Table Of Contents
- Introduction = 1
Materials and Methods = 8
1. Bacterial strain and culture conditions = 8
2. Preparation of genomic DNA from S. iniae = 9
3. Polymerase chain reaction (PCR) amplication of 4-genes = 9
3.1. PCR amplification for full sequencing of GAPDH and α-enolase = 9
3.2. PCR amplification for full sequencing of streptolysin S, Sag A = 10
3.3. PCR amplification for full sequencing of iron uptake ABC transporter, piaA = 10
3.3.1. PCR cloning of partial genomic DNA using degenerated primer = 10
3.3.2. Primer walking of genomic DNA = 11
4. Cloning and sequence analysis = 12
5. Recombinant protein production and purification = 12
5.1. Polymerase chain reaction (PCR) amplification assay and sequencing = 12
5.2. Construction of recombinant protein expression vector = 13
5.2.1. Construction of recombinant protein expression vector, pET 28a = 13
5.2.2. Construction of recombinant protein expression vector, pGEX-4T-1 = 13
5.3. Expression and purification of recombinant proteins = 13
5.3.1. Expression and purification of His-tagged fusion protein = 14
5.3.2. Expression and purification of GST fusion protein = 14
6. Antibody production = 15
6.1. Production of Ab(Antibody) from rat against recombinant proteins = 15
6.2. Bactericidal activity of serum = 16
7. Extraction of cell wall proteins = 16
7.1. Western blot = 16
8. Immunoblot analysis for plasmin(ogen) binding = 17
9. Display the 4 genes on the surface of E. tarda ghost = 18
9.1. PCR amplification for pHCE-InaN-4genes-ghost 37 SDM = 18
9.2. Construction of surface expression vector = 18
9.3. Transformation of bacteria = 19
9.3.1. Transformation of E. coli DH5α by chemical method = 19
9.3.2. Transformation of E. tarda FSW910410 by electroporation = 19
10.Induction of target genes expression and preparation of bacterial ghosts = 19
11. Isolation of outer membrane = 20
11.1. Western blot analysis = 20
12. Statistical analysis = 21
Result = 31
1. Cloning and sequence analysis = 31
1.1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) = 31
1.2. α-enolase = 34
1.3. Streptolysin S-SagA = 39
1.4. piaA = 42
2. Recombinant protein purification and Western blot analysis = 47
2.1. GAPDH = 47
2.1.1. Recombinant protein production and purification = 47
2.1.2. Production of Ab against recombinant protein in the immunized rat = 47
A. Western blot = 47
B. Bactericidal activity = 47
2.2. α-enolase = 51
2.2.1. Recombinant protein production and purification = 51
2.2.2. Production of Ab against recombinant protein in the immunized rat = 51
A. Western blot = 51
B. Bactericidal activity = 51
2.3. Streptolysin S-SagA = 55
2.3.1.Comparison of SagA & SagA-toxic recombinant protein expression and purification = 55
2.3.2. Production of Ab against recombinant protein in the immunized rat = 55
A. Western blot = 55
B. Bactericidal activity of serum = 55
2.4. piaA = 60
2.4.1. Recombinant protein production and purification = 60
2.4.2. Production of Ab against recombinant protein in the immunized rat = 60
A. Western blot = 60
B. Bactericidal activity of serum = 60
3. Cell wall association using immunoblot method = 66
3.1. GAPDH = 66
3.2. α-enolase = 66
4. Plasmin(ogen) binding ability using immunoblot method = 69
4.1. GAPDH = 69
4.2. α-enolase = 69
5. Display the 4 genes on the surface of E. tarda ghost = 72
5.1. Generation of ghost bacteria = 72
5.2. Efficiency of E. tarda ghost generation = 72
5.3. Expression of 4-proteins = 72
Discussion = 78
요약 = 86
참고문헌 = 89
- Degree
- Master
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