Production of rat polyclonal antisera and chicken egg yolk antibody (IgY) to the Escherichia coli-produced recombinant nucleoprotein (NP) of Avian Influenza Virus A/H5N1 type and expression of antigenic NP gene in plant : 조류인플루엔자 A형/H5N1 바이러스 핵단백질 유전자의 식물 및 대장균 발현과 항체생산원문보기
항원 도메인을 갖는 조류인플루엔자바이러스의 핵단백질(NP) 유전자의 일부분을 박테리아 발현 벡터인 pET28에 삽입하여 pET28a/H5N1-NP를 구성하였다. 재조합단백질 NP는 잘 발현되어 세포질에 축적되었기 때문에 Ni-NTA resin을 이용하여 크로마토그래피를 수행하였다. 분리정제된 NP를 면역원으로 실험용 쥐와 닭에 주사한 후 각각의 동물에서 다클론 IgG와 IgY를 획득하여 Western blot을 실시한 결과 양성 상호반응을 보였다. 그러므로 NP 유전자를 식물발현 벡터로 도입하여 발현카세트(Cauliflower mosaic virus (CaMV) 35S ...
항원 도메인을 갖는 조류인플루엔자바이러스의 핵단백질(NP) 유전자의 일부분을 박테리아 발현 벡터인 pET28에 삽입하여 pET28a/H5N1-NP를 구성하였다. 재조합단백질 NP는 잘 발현되어 세포질에 축적되었기 때문에 Ni-NTA resin을 이용하여 크로마토그래피를 수행하였다. 분리정제된 NP를 면역원으로 실험용 쥐와 닭에 주사한 후 각각의 동물에서 다클론 IgG와 IgY를 획득하여 Western blot을 실시한 결과 양성 상호반응을 보였다. 그러므로 NP 유전자를 식물발현 벡터로 도입하여 발현카세트(Cauliflower mosaic virus (CaMV) 35S 프로모터 a TEV 선도염기, pET28a/H5N1-NP로부터 합성한 PCR 산물, 35S 터미네이터)를 구성하였다. 재조합단백질 NP의 발현을 탐색하기 위해 재조합벡터를 함유한 아그로박테리아를 Nicotiana benthamiana의 2개 잎에 주입하였고 (agro-infiltration), 또한 형질전환체를 생산하기 위해 배지 위에 있는 잎디스크에 감염시켰다. NP 유전자가 제대로 식물에 도입되었는가를 확인하기 위해 아그로박테리아를 주입한 잎을 1-4일 후에 수확하여 제한효소 NdeI으로 절단한 결과 삽입됨을 확인할 수 있었고, NP 유전자 특이적 프라이머를 이용하여 RT-PCR을 실시한 결과 삽입된 유전자가 정상적으로 발현되고 있음을 확인할 수 있었다. N. benthamiana에서 발현된 NP는 Western blot에서 쥐에서 생산된 다클론항체와 양성 상호반응을 보였으나, 발현된 NP의 분자량은 45 kDa으로서 기대한 크기보다 크게 나타났다. 이 결과는 아마도 식물내에서 glycosylation되어 변형된 것으로 추정된다.
항원 도메인을 갖는 조류인플루엔자바이러스의 핵단백질(NP) 유전자의 일부분을 박테리아 발현 벡터인 pET28에 삽입하여 pET28a/H5N1-NP를 구성하였다. 재조합단백질 NP는 잘 발현되어 세포질에 축적되었기 때문에 Ni-NTA resin을 이용하여 크로마토그래피를 수행하였다. 분리정제된 NP를 면역원으로 실험용 쥐와 닭에 주사한 후 각각의 동물에서 다클론 IgG와 IgY를 획득하여 Western blot을 실시한 결과 양성 상호반응을 보였다. 그러므로 NP 유전자를 식물발현 벡터로 도입하여 발현카세트(Cauliflower mosaic virus (CaMV) 35S 프로모터 a TEV 선도염기, pET28a/H5N1-NP로부터 합성한 PCR 산물, 35S 터미네이터)를 구성하였다. 재조합단백질 NP의 발현을 탐색하기 위해 재조합벡터를 함유한 아그로박테리아를 Nicotiana benthamiana의 2개 잎에 주입하였고 (agro-infiltration), 또한 형질전환체를 생산하기 위해 배지 위에 있는 잎디스크에 감염시켰다. NP 유전자가 제대로 식물에 도입되었는가를 확인하기 위해 아그로박테리아를 주입한 잎을 1-4일 후에 수확하여 제한효소 NdeI으로 절단한 결과 삽입됨을 확인할 수 있었고, NP 유전자 특이적 프라이머를 이용하여 RT-PCR을 실시한 결과 삽입된 유전자가 정상적으로 발현되고 있음을 확인할 수 있었다. N. benthamiana에서 발현된 NP는 Western blot에서 쥐에서 생산된 다클론항체와 양성 상호반응을 보였으나, 발현된 NP의 분자량은 45 kDa으로서 기대한 크기보다 크게 나타났다. 이 결과는 아마도 식물내에서 glycosylation되어 변형된 것으로 추정된다.
Partial length of nucleoprotein gene (NP) containing antigenic domain of Avian influenza virus A/H5N1-NP, was constructed in a bacterial expression system using pET28 vector (pET28a/H5N1-NP). The majority of recombinant protein was in the soluble cellular fraction in Eschrichia coli. Therefore, the ...
Partial length of nucleoprotein gene (NP) containing antigenic domain of Avian influenza virus A/H5N1-NP, was constructed in a bacterial expression system using pET28 vector (pET28a/H5N1-NP). The majority of recombinant protein was in the soluble cellular fraction in Eschrichia coli. Therefore, the recombinant NP was purified using Ni-NTA chromatography and its polyclonal IgG from rats and IgY from chicken egg yolk were produced against E. coli-produced recombinant H5N1-NP. Western blot analysis showed that rat antisera and chicken IgY were able to recognize well the recombinant NP used as an immunogen. Therefore, the NP gene was introduced into binary vector containing an expression cassette, Cauliflower mosaic virus (CaMV) 35S promoter, a TEV leader sequence, the PCR-amplified NP gene from pET28a/H5N1-NP and a 35S terminator. To express the recombinant protein NP in Nicotiana benthamiana, Agrobacterium containing the chimera construct was infiltrated into two leaves of N. benthamiana for the transient assay and infected onto leaf discs for the production of transgenic plants. To confirm the presence of NP sequences in N. benthamiana, total genomic DNA was extracted and digested with NdeI. The expected size of NP fragment was detected from 1-4 DPI, and the integration of NP DNA in plant genome was confirmed by Southern blot analysis. In addition, RT-PCR was performed using NP gene-specific primers, resulted in the amplification of expected 801 bp fragments from total RNA which was extracted from agro-infiltrated leaves. All the results taken together verified that the target gene was successfully integrated into the genomic DNA of infiltrated leaf tissues, and suggested that the inserted NP gene is functional at transcriptional level. The recombinant NP expressed in N. benthamiana was well cross-reacted with rat polyclonal antisera against Esherichia coli-produced NP. However, its size was 45 kDa which is higher than the predicted molecular weight of recombinant protein from the cDNA sequence. It could be experienced the protein modification in plant cell, possibly by glycosylation.
Partial length of nucleoprotein gene (NP) containing antigenic domain of Avian influenza virus A/H5N1-NP, was constructed in a bacterial expression system using pET28 vector (pET28a/H5N1-NP). The majority of recombinant protein was in the soluble cellular fraction in Eschrichia coli. Therefore, the recombinant NP was purified using Ni-NTA chromatography and its polyclonal IgG from rats and IgY from chicken egg yolk were produced against E. coli-produced recombinant H5N1-NP. Western blot analysis showed that rat antisera and chicken IgY were able to recognize well the recombinant NP used as an immunogen. Therefore, the NP gene was introduced into binary vector containing an expression cassette, Cauliflower mosaic virus (CaMV) 35S promoter, a TEV leader sequence, the PCR-amplified NP gene from pET28a/H5N1-NP and a 35S terminator. To express the recombinant protein NP in Nicotiana benthamiana, Agrobacterium containing the chimera construct was infiltrated into two leaves of N. benthamiana for the transient assay and infected onto leaf discs for the production of transgenic plants. To confirm the presence of NP sequences in N. benthamiana, total genomic DNA was extracted and digested with NdeI. The expected size of NP fragment was detected from 1-4 DPI, and the integration of NP DNA in plant genome was confirmed by Southern blot analysis. In addition, RT-PCR was performed using NP gene-specific primers, resulted in the amplification of expected 801 bp fragments from total RNA which was extracted from agro-infiltrated leaves. All the results taken together verified that the target gene was successfully integrated into the genomic DNA of infiltrated leaf tissues, and suggested that the inserted NP gene is functional at transcriptional level. The recombinant NP expressed in N. benthamiana was well cross-reacted with rat polyclonal antisera against Esherichia coli-produced NP. However, its size was 45 kDa which is higher than the predicted molecular weight of recombinant protein from the cDNA sequence. It could be experienced the protein modification in plant cell, possibly by glycosylation.
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