흰반점바이러스(WSSV)의 중화를 위한 재조합단백질 rVP466의 항혈청 생산 Production of the Antiserum against Recombinant Envelop Protein, rVP466 for the Neutralization of White Spot Syndrome Virus (WSSV)원문보기
본 연구는 WSSV의 재조합단백질 rVP466에 대하여 생산된 항혈청을 사용하여 WSSV에 대한 neutralization (중화) 효과를 확인하고자 수행하였다. 먼저 재조합단백질 rVP466의 생산을 위해 WSSV의 구성단백질 VP466을 암호화하는 유전자인 VP466을 포함하는 재조합 플라스미드 pCold-VP466을 제작한 다음 이것을 발현용 숙주인 E. coli RIPL에서 발현하였다. 발현된 rVP466에 대한 항혈청은 토끼를 사용하여 생산하였으며, 항원 rVP466에 대한 특이면역반응은 Western blot을 통해 확인하였다. WSSV에 대한 항혈청의 중화효과를 확인하기 위해 항혈청과 반응시킨 바이러스액($1{\times}10^4$ 배로 희석된 WSSV)을 이용하여 실험용 새우(Penaeus chinensis)에게 주사 감염을 통해 공격실험(challenge test)을 수행하였다. 실험 결과, WSSV로 공격실험한 감염대조구(positive control)의 새우들은 감염 후 17일째에 100% 누적폐사율을 보였으며, preimmune serum과 WSSV의 혼합액을 challenge한 preimmune control의 새우들은 감염 후 25일째에 83%의 누적폐사율을 보였다. WSSV와 rVP466 항혈청을 1:0.01, 1:0.1, 1:1로 혼합한 액으로 challenge한 새우들은 감염 후 25일째에 각각 73%, 53%, 46%의 누적폐사율을 보였다. 이상의 결과를 통해 WSSV가 rVP466 항혈청에 의해 농도의존적으로 neutralization됨을 확인하였으며, 이는 WSSV 감염과정에 VP466이 관여함을 나타내는 것이다.
본 연구는 WSSV의 재조합단백질 rVP466에 대하여 생산된 항혈청을 사용하여 WSSV에 대한 neutralization (중화) 효과를 확인하고자 수행하였다. 먼저 재조합단백질 rVP466의 생산을 위해 WSSV의 구성단백질 VP466을 암호화하는 유전자인 VP466을 포함하는 재조합 플라스미드 pCold-VP466을 제작한 다음 이것을 발현용 숙주인 E. coli RIPL에서 발현하였다. 발현된 rVP466에 대한 항혈청은 토끼를 사용하여 생산하였으며, 항원 rVP466에 대한 특이면역반응은 Western blot을 통해 확인하였다. WSSV에 대한 항혈청의 중화효과를 확인하기 위해 항혈청과 반응시킨 바이러스액($1{\times}10^4$ 배로 희석된 WSSV)을 이용하여 실험용 새우(Penaeus chinensis)에게 주사 감염을 통해 공격실험(challenge test)을 수행하였다. 실험 결과, WSSV로 공격실험한 감염대조구(positive control)의 새우들은 감염 후 17일째에 100% 누적폐사율을 보였으며, preimmune serum과 WSSV의 혼합액을 challenge한 preimmune control의 새우들은 감염 후 25일째에 83%의 누적폐사율을 보였다. WSSV와 rVP466 항혈청을 1:0.01, 1:0.1, 1:1로 혼합한 액으로 challenge한 새우들은 감염 후 25일째에 각각 73%, 53%, 46%의 누적폐사율을 보였다. 이상의 결과를 통해 WSSV가 rVP466 항혈청에 의해 농도의존적으로 neutralization됨을 확인하였으며, 이는 WSSV 감염과정에 VP466이 관여함을 나타내는 것이다.
This study was carried out to evaluate neutralization effects against WSSV using antiserum produced from recombinant envelop protein, rVP466 of WSSV. The VP466 gene of WSSV was cloned into pCold I expression vector and rVP466 was expressed in E. coli RIPL. The antiserum against rVP466 was produced i...
This study was carried out to evaluate neutralization effects against WSSV using antiserum produced from recombinant envelop protein, rVP466 of WSSV. The VP466 gene of WSSV was cloned into pCold I expression vector and rVP466 was expressed in E. coli RIPL. The antiserum against rVP466 was produced in white rabbits (New Zealand white rabbit). The specific immunoreactivity to the antigen, rVP466, was confirmed by Western blot. The constant amounts of WSSV at $1{\times}10^4$ diluted stocks were mixed with various antiserum concentrations and then injected to the muscle of shrimp, Penaeus chinensis, for the neutralization challenge. The shrimps challenged with WSSV as a positive control and those with the mixture of WSSV and preimmune serum as a preimmune control showed 100% cumulative mortality at 17 days post challenge and 83% at 25 days post challenge, respectively. The shrimps challenged with 3 different mixtures of WSSV and rVP466 antiserum at ratios of 1:0.01, 1:0.1 and 1:1 showed 73%, 53% and 46% cumulative mortalities at 25 days post challenge, respectively. These results indicated that WSSV could be neutralized by the rVP466 antiserum. These results suggest that envelop protein VP466 is involved in the initial step of WSSV infection in shrimp.
This study was carried out to evaluate neutralization effects against WSSV using antiserum produced from recombinant envelop protein, rVP466 of WSSV. The VP466 gene of WSSV was cloned into pCold I expression vector and rVP466 was expressed in E. coli RIPL. The antiserum against rVP466 was produced in white rabbits (New Zealand white rabbit). The specific immunoreactivity to the antigen, rVP466, was confirmed by Western blot. The constant amounts of WSSV at $1{\times}10^4$ diluted stocks were mixed with various antiserum concentrations and then injected to the muscle of shrimp, Penaeus chinensis, for the neutralization challenge. The shrimps challenged with WSSV as a positive control and those with the mixture of WSSV and preimmune serum as a preimmune control showed 100% cumulative mortality at 17 days post challenge and 83% at 25 days post challenge, respectively. The shrimps challenged with 3 different mixtures of WSSV and rVP466 antiserum at ratios of 1:0.01, 1:0.1 and 1:1 showed 73%, 53% and 46% cumulative mortalities at 25 days post challenge, respectively. These results indicated that WSSV could be neutralized by the rVP466 antiserum. These results suggest that envelop protein VP466 is involved in the initial step of WSSV infection in shrimp.
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제안 방법
coli. After the cloning, digestions of pCold-VP466 were carried out with BamH I and Hind III and the restriction map was prepared as shown in Fig. 1. The 1381 bp fragment of the VP466 gene was observed, indicating correct construction of pColdVP466.
In this study, the productions of antiserum against recombinant envelop protein, rVP466 and neutralization of WSSV was carried out to elucidate infection mechanism of VP466 of WSSV.
The VP466 specific primer sets of forward primer 5’-GGATCCATGTCTGCATCTTTAATATTGGAC-3’ and reverse primer 5’-AAGCTTGTTATGACACAAACCTATTCCACAA-3’ with BamH I and Hind III restriction site, respectively, were prepared based on the nucleotide sequences of VP466.
대상 데이터
Twenty-five microliters of different virus dilutions were injected in the second abdominal segment of the shrimp using a 30-gauge needle. For each group, ten shrimps were used in the challenge test. Mortality was recorded daily and dead shrimps were tested for the presence of WSSV by PCR.
Healthy shrimps (Penaeus chinensis) were purchased from a shrimp farm located in Goheung, Jeonnam, Korea. Shrimps of 6 to 8 grams were used in experiments.
In addition to major proteins, 40 WSSV proteins have been characterized [2]. The VP466 gene is located at 177124-178521 of WSSV genome (Genbank accession no. AF395545). The open reading frame (ORF) of VP466 gene is composed of a 1381 bp fragment encoding a protein of 466 amino acids with a deduced molecular mass of about 50 kDa [6].
Fifty microliter mixtures of WSSV and antiserum were intramuscularly injected into shrimp. The experiments were conducted in triplicate. After the injection, the shrimp mortality was monitored daily.
The virus particle of WSSV consists of 6 major proteins such as VP15, VP19, VP24, VP26, VP28 and VP664. In addition to major proteins, 40 WSSV proteins have been characterized [2].
성능/효과
For the determination of dilution ratio resulting in 100% mortality of shrimp for desired period of experiments, in vivo virus titration was performed. As shown in Fig. 3, the results of in vivo titration showed 50-100% mortalities in all tested groups at 14 days of post challenge. Administration of virus dilutions of 1×101, 1×102, 1×103 and 1×104 resulted in 100% mortality in less than 11 days of post challenge.
Based on these results, the recombinant envelop protein rVP466 from WSSV genomic DNA and the antiserum raised against rVP466 were successfully produced and the rVP466 antiserum had a proper immune reactivity with the antigen, rVP466.
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