Immunization and Safety of Recombinant Respiratory Syncytial Virus Fusion Protein TB10.

4-F1
Author :WangRuiBo
Tutor:JingShenRong
School :Kunming University of Science and Technology
CLC :R725.6
TYPE :Masters thesis
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Year:2011
Abstract:
Respiratory syncytial virus (RSV) is the most major pathogens of lower respiratory tract infection in
infants worldwide, which are likely to cause bronchiolitis, pneumonia, or death. There is no useful
vaccine to prevent RSV infection. In this study, the F1 protein of RSV, fused with the TB10.4 proteins
of Mycobacterium tuberculosis was expressed in E. coli, and immunised BALB/c mice combined with
mucosal adjuvants non-toxic E. coli heat-labile enterotoxin (LT). Finally, we evaluated the
immunogenicity and safty of the fusion protein.We obtained the recombinant fusion protein TB10.4F1 and protein TB10.4, which contain His-tag in E. coli, by constructed the recombinant vector of
TB10.4-F1/pET30a and TB10.4/pET28a, and proved by western-blot with anti-His tag antibody. The
recombinant inclusion bodies protein was purified with nickel affinity chromatography under
denaturing conditions. The purity of TB10.4-F1 and TB10.4 was 80% and 95%, respectively.The
purified TB10.4-F1 and TB10.4 were immunised BALB/c mice combined with LT. Twenty five mice
were divided into 5 groups, each group were immunized at week 0,2,4 as the following:PBS group; LT
group; TB10.4 group; TB10.4-F1 group; TB10.4-F1+LT group. The results showed that:the specific
IgG of TB10.4-F1+LT group and TB10.4-F1 group were higher than other groups obviously, which
suggested that fusion protein could induce a high level of serum antibody;compared with the
TB10.4 group, the IgG1/IgG2a ratio of TB10.4-F1+LT group and TB10.4-F1 group was closer to 1,
indicating that T cell immune response was more balanced;TB10.4-F1 fusion protein had immune
toxicity in lung biopsy, which can cause lung telangiectasia, eosinophilia;the IgG1/IgG2a ratio of
TB10.4 group was greater than 1, which represented that the Th2-type response is dominant.Mice
were challenged with 3105 pfu of RSV by intranasally after immunization.5 days later, mice were
killed for detection. The results suggeted that:mice weight of TB10.4-F1+LT group and TB10.4-F1
group decreased quickly, three mice in TB10.4-F1+LT group were dead due to respiratory
failure;the douche of the respiratory tract of TB10.4-F1+LT group and TB10.4-F1 group had a good
IgA response, and TB10.4-F1+LT group was higher than TB10.4-F1 group (P<0.05), indicating that
LT can promote production of secretory IgA on the respiratory mucosa;lung biopsy showed that
TB10.4-F1+LT group and TB10.4-F1 group were induced serious Immune toxicological reaction after
challenge, indicating that the recombinant protein TB10.4-F1 not only failed in protection, but
increased the disease in mice.This study investigated the immunogenicity and safety of fusion protein
TB10.4-F1,which will provide a theoretical basis for developing new RSV vaccines.