Development and in vitro expression of the recombinant monoclonal antibody against grouper nervous necrosis virus

• Main Authors: Jui-Shin Chang, 張瑞昕
• Other Authors: Shau-Chi Chi
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/35673236655608790211

碩士 === 國立臺灣大學 === 動物學研究所 === 97 === The aquaculture production of marine fish is an important economic industry in Taiwan. One of the highly value marine fish is groupers. However, the occurrence of viral nervous necrosis disease persists as a stumbling block that besets sustainable production in grouper larvae. Nervous necrosis virus (NNV) is the causative agent of mass mortality of cultured grouper at larval stage. It belongs the betanodavirus of Nodaviridae, with 2 segments of single strand positive sense RNA. The immune system of grouper larvae are not mature enough to be immunized until 20-30 days post hatchery (d.p.h.). Therefore, the larvae are very sensitive to NNV infection within 30 d.p.h. Passive immunization was therefore an alternative prophylaxis strategy against NNV infection at this stage. Five NNV neutralization monoclonal antibodies (mAb) are developed in our previous study. The mAb 9D exhibits high neutralizing activity and recognizes linear epitope of capsid protein. In the present study, the heavy and light chain genes of hybridoma cells of mAb 9D were cloned. The full length of these two genes were determined by 5’ and 3’ RACE PCR, and then inserted into the expression vector, respectively. The constructed vectors were transfected into GF-1 cells. The mRNA of heavy chain and light chain genes were detected by RT-PCR, and the antibody proteins expressed in the transfected cells were detected by western blot. Moreover, the expressed mAb 9D in the culture supernatant of transfected cells was confirmed to recognize NNV capsid protein by western blot. In the future, the constructed mAb may be applied in the passive immunity of grouper larvae in order to decrease threaten of VNN disease.