PEG-modified subunit vaccine encoding dominant epitope to enhance immune response against spring viraemia of carp virus.

Spring viraemia of carp (SVC) caused by spring viraemia of carp virus (SVCV) can infect almost all fish of cyprinids, which bring huge economic losses to aquaculture. Glycoprotein (G), as the most important antigenic determinant protein of SVCV, is widely considered as an effective method against SVCV. In our previous study, we found that G3 (131 aa) is the potential dominant antigen epitope that induces strong immune responses similar to G protein (510 aa). Here, in order to further improve the immune effect, we reported a subunit vaccine (PEG-G3) constructed by PEG-modified dominant epitope protein (G3). The results of serum antibody production, enzyme activities and immune-related genes expression showed that PEG-G3 induces significantly stronger immune protective responses against SVCV than G3. PEG modification significantly increased the serum antibody level of the vaccine, which increased significantly after immunization and reached the peak at 21 day post-vaccination. T-AOC and AKP activities in the lowest concentration group (5 µg) of PEG-G3 were significantly higher than those in the highest concentration group (20 µg) of G3. In PEG-G3 group, the expression of almost all genes increased at least 4 times compared with the control group. After 14-day challenge, the RPS (relative percentage survival) of the highest concentration of PEG-G3 group was 53.6%, while that of G3 group is 38.9%. Therefore, this work shows that PEG modification and dominant epitope screening may be effective methods to improve the immune protective effect of vaccines and to resist the infection of aquatic animal viral diseases.

[Influence of penetrative needling of Shendao (GV 11) on the symptom score and serum IgE content in chronic urticaria patients].

OBJECTIVE: To observe the changes of symptom scores and serum IgE level after treatment with thick-needle subcutaneous penetration of Shendao (GV 11) in chronic urticaria patients. METHODS: A total of 60 chronic urticaria patients were randomly divided into acupuncture group (n = 30) and medication group (n = 30). Subcutaneous penetrative needling was applied to GV 11 with thick acupuncture needle (retained for 4 h/time, once daily, 5 times/week) for patients of acupuncture group and Levocetirizine Hydrochloride tablets (5 mg/time, once daily in the first two weeks, then, once every other day in the 3rd and 41th weeks, and once every 3 days in the last two weeks) were given to patients of medication group. Serum IgE content was assayed before and 2,6, 12 weeks after the treatment by chemiluminescent technique. Symptom scores were obtained by "0-3 four levels assessment" method in the light of the size of the wheal and the itching severity. RESULTS: Self-comparison indicated that the symptom scores and serum IgE levels declined significantly (P < 0.01) 2 and 6 weeks (Wks) after the treatment in both acupuncture and medication groups,and 12 Wks after the treatment in the acupuncture group (P < 0.01). Comparison between two groups showed that the symptom score and serum IgE content of acupuncture group were significant lower than those of medication group 12 Wks after the treatment (P < 0.05). No significant differences were found between two groups in the symptom scores and serum IgE levels before, 2 and 6 Wks after the treatment (P > 0.05). A positive correlation exists between the symptom score and the serum IgE level before and after the treatment in both groups. CONCLUSION: Thick-needle subcutaneous penetration of Shendao (GV 11) can effectively improve clinical symptoms of chronic urticaria patients, which may be closely related to its effect in lowering peripheral blood IgE level.

[Cloning of the variable region genes from hybridoma against bFGF and expression of single chain antibody fragments in E.coli HB2151].

AIM: To construct and express the single chain antibody (scFv) in E.coli HB2151 by cloning the variable region genes from hybridoma against bFGF. METHODS: Total RNA was extracted from hybridoma cell line B2F3 secreting mAbs against bFGF and the cDNA was amplified by retropolymerase chain reaction (RT-PCR). V(L) and V(H) were fused by a short peptide linker containing 15 amino acids (Gly(4)Ser)(3) using splice-overlap extension PCR to construct the scFv gene. The sequences of the scFv were analyzed by Shanghai Sangon Biological Engineering Technology and Services Co. Ltd and Ig Blast data base in GenBank. The scFv gene was inserted into pCANTAB-5E vector and expressed in E.coli HB2151. RESULTS: The V(H) gene contained 375 base pairs and encoded 125 amine acid residues. The V(L) gene contained 399 base pairs and encoded 133 amine acid residues. There were four FRs, three CDRs and two characteristic cysteine residues in the V(H) gene and the V(L) gene, respectively. The scFv gene contained 789 base pairs and encoded 263 amine acid residues with the structure of V(H)-linker-V(L). Restriction endonuclease digestion and DNA sequencing proved that the expression vector of pCANTAB-5E-scFv was constructed correctly. SDS-PAGE and ELISA analysis showed that scFv was successfully expressed in E.coli HB2151 and the expression protein had specific antigen binding activity. CONCLUSION: The variable region genes of anti-bFGF mAbs have been cloned successfully and single chain antibody fragments have been constructed and expressed, which will be a great help to the study of humanized antibodies against bFGF.