Construction Of Infectious Hematopoietic Necrosis Virus(IHNV) Glycoprotein Recombinant Chlamydomonas Reinhardtii And A Preliminary Study Immunological Propertiesexperiment Research

Infectious hematopoietic necrosis is composed of infectious hematopoietic necrosis virus caused by an acute,systemic disease of fish,the main object of infection is cold water fish such as salmon,trout and so on.In 1985,the disease broke out in the district,Benxi City,Liaoning province,China,and caused huge economic losses to aquaculture.When the fish ill body hemorrhage,kidney and spleen hematopoietic tissue necrosis is typical features,IHN infection caused by adult fish mortality was 10~25%,and the larval mortality rate could reach 100%. Office International Des Epizooties made it as a must declare animal epidemics,China is displayed it in two class of infectious diseases directory,also is the first class port fish quarantine object.Therefore to develop a new vaccine have good immune effect,high safety,low cost characteristic become a research hotspot.At present,using plants as bioreactors,to produce bio-pharmaceutical development of transgenic plants is widely interest by scientists.Chlamydomonas reinhardtii as a new plant expression system,not only can accurately express the exogenous gene,good production environment security general advantage,but also has advantages of the chlamydomonas reinhardtii cell structure is simple,easy culture conditions,propagation speed,can be mass production.At the same time also has oneself characteristics that is advantages of low cost, photosynthetic efficiency high.Using it as a bioreactor for fish developed an oral vaccine has a good prospect. Algae is the natural food of fish,using Chlamydomonas reinhardtii to studying IHNV has a unique advantage.This study will IHNV replication in FHM monolayers cell,collect cell and viral when showed typical CPE.Extracting total RNA from cells and IHNV,with the total RNA as the template for RT-PCR,to obtain G protein gene.The G protein gene was cloned into pMD-18 T vector,recombinant plasmid pMD-18T-IHNV-G was successfully constructed.Plasmid amplification by TOP10 E.coli,extraction of plasmid.With double enzyme method validation and genetic sequencing,sequencing results show homology is 99% with IHNV HLJ-09 strain(accession number:JX649101).Recombinant plasmid pMD-18T-IHNV and pChlamy-4-G vector recovery after double enzyme,connected them by T4 ligase,then constituted recombinant plasmid p Ch-IHNV-G.Amplification recombinant plasmid pCh-IHNV-G by TOP10 E.coli,using PCR validation plasmid,then linearized recombinant plasmid,using electroporation technology import it into chlamydomonas reinhardtii that cell wall deficiency.Using bleomycin screened,extraction DNA from transgenic Chlamydomonas reinhardtii and then PCR,to prove that the IHNV glycoprotein gene has been integrated into the nuclear genome of Chlamydomonas reinhardtii.Extraction total protein from transgenic Chlamydomonas reinhardtii,after SDS-PAGE electrophoresis analysis get a protein band about 58 KDa,consistent with the expected results.Western blot analysis showed that the transgenic Chlamydomonas reinhardtii expressed protein can be combined with 6×HIS tag antibody specificity,has good reactogenicity.The transgenic Chlamydomonas reinhardtii,Chlamydomonas reinhardtii,PBS solution were respectively fed to the Balb/c,continuous lavage 7 days then challenged with IHNV,record mice body weight on day 0,7,14,21,28.7days after challenge,take the mouse spleen,preparation of spleen cell suspension,flow cytometric analysis of T cell subsets.From body weight change in mice found when Chlamydomonas reinhardtii fed mice weight decreased compared with the control,but weight gain obvious than others after challenge.Flow cytometry results show that CD4+,CD8+T lymphocytes content increased in transgenic Chlamydomonas reinhardtii group.All showed that the transgenic Chlamydomonas reinhardtii can stimulate T lymphocytes value added,which is helpful to improve the immune response.This study shows that transgenic chlamydomonas reinhardtii with IHNV glycoprotein gene can stimulate the appreciation of T cells in the body,help to improve the body’s immune function. Transgenic Chlamydomonas reinhardtii have developed an oral vaccine precondition,to lay a solid foundation for the further development of efficient and safe oral fish vaccine or can improve immune function mix in the feed.