Oral Subunit Vaccine VP28against WSSV And Promoting The Growth Of Crayfish Oral DNA-drug

White spot syndrome virus（WSSV） is a highly pathogenic virus that infectsshrimp and other crustaceans. Since the first report of WSSV in Taiwan in1993, it hasrapidly spread to all the major shrimp farming areas in Japan, Southeast Asia, NorthAmerica and other countries. It resulted in a great loss annually. Researchers try tofind effective strategies to prevent and control WSSV. So far there’s been no effectiveproduct or practical measure to deal with WSSV.HIV-1Tat protein is a transcriptional activator. The study found that the proteindomain structure, including11amino acid peptide sequence, the short peptides coulddeliver heterologous proteins across most biomembranes without losing bioactivity.TAT protein transduction domain as a delivery vector in mice, rabbits, mammaliancells, insect cells were studied extensively, but has not been reflected for thecrustaceans-Crayfish.In this study we constructed recombinant plasmids and transformed them intoE.coli strain BL21(DE3) to express TAT-VP28, VP28, TAT-VP28-EGFP andVP28-EGFP fusion proteins. Crayfish in experimental groups were individually fedthe same amount of purified proteins (TAT-VP28-EGFP, VP28-EGFP) via a feedingtube. Immunohistochemistry and flow cytometry methods confirmed that TAT-fusionproteins are able to transduce across shrimp the intestinal wall tissue and go intohemolymph. Strong green-fluorescent signals of EGFP were detected in the inner layer of the mid gut, muscle and heart sections from crayfish fed withrTAT-VP28-EGFP.The purified proteins VP28and TAT-VP28were used to orally immunize C.clarkii.The supernatants of their hemolymph were collected and processed individually toassay the activities of phenoloxidase (PO) and superoxide dismutase (SOD). It wasfound that the rTAT-VP28resulted in the most pronounced increase of PO and SODactivity after2hours immunization. After immunization rVP28, the PO enzymeactivity did not change, SOD activity increase after24hours. This result once againconfirms that TAT-fusion protein are able to transduce across shrimp the intestinalwall tissue and go into hemolymph, the shrimp can cause the immune response.To evaluate the potential of the subunit vaccine, vaccination experiment wasconducted after shrimps were fed for7and14days respectively. The pelletscontained a daily dose of100μg rVP28and100μg rTAT-VP28per shrimp. Thechallenge test was conducted with an injection of100μl WSSV at7and14dayspost-last feeding. The results showed that with the positive control group at10daysmore than90%mortality.TAT-VP28, VP28group the mortality was43.3%,36.7%;60%,50.0%. In the vaccination experiments, TAT-VP28, VP28and control groupsafter the14days of vaccination, which were subsequently challenged at3and7dayspost vaccination. The results showed that with the positive control group at10daysmore than90%mortality.TAT-VP28, VP28group the mortality was43.3%,53.3%;60%,66.7%.Our study here demonstrates, for the first time, the C. clarkia is as an animal model,we attempted to utilize the Tat-derived peptide as an oral delivery method for subunitvaccine against WSSV in C. clarkia. TAT-fusion proteins are able to transduce acrossshrimp gut and go into hemolymph and other tissues. After immunization, it wasfound that the rTAT-VP28resulted in the most pronounced increase of PO and SODactivity. So it enhanced the immune defense system of shrimp. The study provides anew way to design more practical strategies for prevent and control WSSV. Fish growth hormone(fGH)is a polypeptide produced by the pituitary cells ofteleosts, which involved in protein synthesis and lipid metabolism and otherphysiological functions in fish. fGH has been demonstrated to promote growth in fish,it has high economic value in aquaculture areas. Electroporation of Litopenaeusschmitti embryos was used to transfer the plasmid that contains the tilapia growthhormone gene(tiGH), the plasmid was introduced shrimp embryos fertilized eggs, thefollow-up study confirmed that compared with the control group, juvenile shrimpweight increase of32%.Live attenuated attenuated Salmonella typhimurium have been used as a deliverysystem for DNA vaccines in vertebrate and invertebrate such as mice, poultry, fishand shrimp. The signal peptide of Penaeidin-2and common carp growth hormonegene fusion construct into the pcDNA3.1-. The plasmid was transferred into COS-7,using RT-PCR and Western blot methods confirmed SGH was highly expression.Crayfish were injected intramuscularly with pcDNA3.1-, pcDNA3.1-SGH, usingreal-time fluorescence quantitative method to detect the levels of transcription ofAstacidin; Chymotryspin and Trypsin in hemolymph. The levels of transcription ofAstacidin; Chymotryspin and Trypsin in hemolymph are increased in pcDNA3.1-SGH group comparing to pcDNA3.1-group. So juvenile crayfish were increased more than22.2%body weigh in25days.The plasmid pcDNA3.1-and pcDNA3.1-SGH were transformed to SV4089byelectroporation and the produced recombinant bacteria were named SV/pcDNA3.1-and SV/pcDNA3.1-SGH, respectively. Recombinant bacteria and recombinantSV/pcDNA3.1-SV/pcDNA3.1-SGH coated feed, to109CFU/daily dose ofcontinuous feeding crayfish juveniles ten days. To investigate whether S.Typhimurium strain SV4089could orally deliver the plasmid pcDNA3.1-SGH intocrayfish and how long the recombinant bacteria could exist in the crayfish,hepatopancreas, swimming foot, gills, heart, hepatopancreas and tail muscle werecollected for isolation of the recombinant bacteria SV/pcDNA3.1-SGH at1,3,7,10and14days post oral administration. For RNA extraction, results in the above tissuesdetected gh transcription. Juvenile crayfish were increased more than21%bodyweigh in one month.This is the first report of the oral growth hormone oral DNA vaccine, confirming theattenuated Salmonella typhimurium DNA vaccine plasmid to transfer to the crayfishbody. It provides a new way to design more practical strategies for the growth ofjuvenile crayfish.