| Hepatitis E (hepatitis E, HE) is caused by hepatitis E virus (hepatitis E, virus, HEV) a digestive tract caused by the spread of an infectious disease, often caused by outbreaks due to faecal contamination of water supplies, the major clinical manifestations of acute hepatitis in pregnant women, the mortality rate is up to 20%, and HEV in patients with chronic liver diseases the infection can lead to liver failure. In addition, in recent years found in organ transplantation and in patients with immunodeficiency hepatitis E can be chronic and lead to liver fibrosis. Hepatitis E is more common in developing countries, but because of animal origin and characteristics of HEV, in recent years in developed countries, the incidence has increased significantly, China has more than the number of hepatitis A incidence in number in 2012 the incidence of hepatitis E, has become a serious public health problem. Therefore, strengthen the research and development of vaccine against hepatitis E virus and timely application of great significance on the prevention and control of hepatitis E.HEV is a non enveloped RNA virus, genome, containing ORF1, ORF2 and ORF3 three. The reading frame of ORF2 encoding structural proteins (pORP2) which contains 660 amino acids (amino, acid, AA) located on the surface of the virus, a virus capsid. Our previous study found that the recombinant protein HEV of ORF2 gene encoding protein C 452-617aa (p166) containing the HEV configuration dependent neutralizing epitopes, the major epitope is HEV, so p166 can be used as coating antigen, anti -HEV. based application of indirect method to detect serum ELISA in our use of Escherichia coli expressed HEV containing neutralizing epitope of the soluble truncated pORF2 protein p179 (aa439-617), found that p179 can effectively induce high titers of neutralizing antibody to HEV, can protect the animal against HEV attacks, can be used for the development of hepatitis E vaccine. The recombinant p179 vaccines have entered clinical trials stage.This paper is in the objective, namely protein on the basis of the study were N-terminal and C-terminal extension, constructed the three contains more antigen epitopes of HEV ORF2 truncated protein and studies on the immunogenicity and antigenicity, and aims to provide a more scientific basis for the further research of recombinant hepatitis E vaccine. Research results include the three parts as follows:1.Truncated protein pORF2aa439-660, pORF2aa422-637, pORF2aa430-660 expression and characterizationDue to the protein can effectively express is hard to predict, early experiments to namely based were designed 20 different length of ORF2 truncated fragments were expressed. Using gene recombination technique construct containing plasmid pET28a recombinant HEV ORF2, and expressed in Escherichia coli, the 13 truncated proteins can be expressed efficiently and these proteins were named for pORF2aa439?627, pORF2aa439?637, pORF2aa439?647, pORF2aa439?660, pORF2aa422?617, pORF2aa422?627, pORF2aa422?637, pORF2aa430?617, pORF2aa430?647, pORF2aa430?660, pORF2aa402?606, pORF2aa410?606 and pORF2aa419?606.13 kinds of protein expression in the success, we have selected the longer segment of the three recombinant proteins pORF2aa439-660, and pORF2aa430-660 pORF2aa422-637 of soluble protein analysis, found that these 3 kinds of different length of proteins can exist in the bacterial lysate supernatant, presenting soluble expression analysis of.SDS-PAGE display respectively pORF2aa439-660, pORF2aa422-637 and pORF2aa430-660 molecular mass of 24.4kDa, and 25.4kDa 23.8kDa. Under the natural state is a dimer. Finally according to the expression of protein carrying his tag, using metal ion affinity chromatography technology won the purified protein.2. The truncated protein pORF2aa439-660, pORF2aa422-637, pORF2aa430-660 and p179, p166 a comparative study of the immunogenicity and antigenicityIn order to further study the 3 different lengths of truncated proteins with p166, the difference of p179 immunogenicity and antigenicity of 5 HEV recombinant protein was purified after diluted with saline to 100mg/L, and then add the volume of aluminum adjuvant (1g/L), reversed mixing for the preparation of the 30min, i.e. the experimental method of intradcrmal injection of hepatitis E vaccine. The 5 groups were immunized mice,6 rats in each group, each of 200 L (antigen containing 10g), and in third weeks after the first immunization, fifth weeks and 33 weeks respectively. The 1 immunization, immunization dose and the same way with the first immunization regularly. Mice serum was collected, respectively p166, p179, pORF2aa439?660, pORF2aa422?637, pORF2aa430?660 as coating antigen to detect p166, p179, pORF2aa439-660, anti-HEV IgG antibody in the serum of pORF2aa422-637 and pORF2aa430-660 after immunization, observed increased antibody titer maintained, decreased until the change process After the first immunization 35 weeks, mice in the same group of each a serum at the same dilution OD value averaged statistics. The results are as follows:A. After the first immunization for 3 weeks,5 weeks,7 weeks, the 15 week, week 35 and 31, the objective from immune rats serum with objective packet is detected, anti HEV IgG antibody titers were 1:1600,1:6400,1:51200,1:25600,1:3200,1:51200; namely, pORF2aa439?660 pORF2aa422?637, pORF2aa430?660 packet detection plate, the antibody titers of the same, respectively for 1:800,1:3200,1:25600,1:12800,1:3200,1:25600; B. First immune after 3 weeks,5 weeks,7 weeks, the 15 week, week 35 and 31, namely from immune rats serum objective detection, anti HEV IgG antibody titers respectively 1:3200,1:12800,1:51200,1:25600,1:6400,1:51200; Namely, pORF2aa439-660 pORF2aa422-637, pORF2aa430-660 packet detection board, the obtained antibody titer of the same, are respectively for 1:1600,1:12800,1:51200,1:12800,1:6400,1:51200; C. After the first immunization for 3 weeks,5 weeks,7 weeks, the 15 week, week 35 and 31, pORF2aa439-660 from immune rats serum with objective detection, anti HEV IgG antibody titers respectively 1:12800,1:409600,1:1638400,1:204800,1:204800,1:1638400; namely detection and antibody titer points 1:3200,1:102400,1:819200,1:102400,1:204800,1:1638400; detected by pORF2aa439?660 and antibody titer of respectively1:6400,1:102400,1:409600,1:204800,1:204800,1:819200; detected by pORF2aa422?637 and antibody titers were for 1:3200,1:102400,1:409600,1:204800,1:204800,1:1638400; with pORF2aa430-660 test, antibody titers were 1:3200,1:102400,1:409600,1:204800,1:204800,1:819200;D. After the first immunization for 3 weeks,5 weeks,7 weeks, the 15 week, week 35 and 31, pORF2aa422-637 from immune rats serum with objective detection, anti HEV IgG antibody titers respectively 1:6400,1:204800,1:819200,1:204800,1:204800,1:1638400; namely detection and antibody titers for 1:3200,1:102400,1:819200,1:204800,1:204800,1:1638400; pORF2aa439-660, pORF2aa422-637. Detection of pORF2aa430?660, the antibody titers of the same are respectively for 1:6400,1:204800,1:819200,1:204800,1:204800,1:819200; E. After the first immunization for 3 weeks,5 weeks,7 weeks, the 15 week, week 35 and 31, pORF2aa430?660 from immune rats serum with objective detection, anti HEV IgG antibody titers respectively 1:6400,1:409600,1:819200,1:204800,1:204800,1:819200;Namely were 1:3200,1:102400,1:409600,1:204800,1:204800,1:819200;pORF2aa439?660,pORF2aa42 2?637. Detection of pORF2aa430?660, the antibody titers of the same are respectively for 1:6400,1:204800,1:409600,1:204800,1:204800,1:409600.Based on the above results, we found that in the same immune time, pORF2aa439?660 pORF2aa422?637, serum titers of pORF2aa430?660 immunized mice significantly higher than objective, namely the serum titer, indicating that the immunogenicity of long protein fragment was better in the short fragment protein. In addition, a serum, in the same titres, with the objective to package is detected income OD value of the average value is greater than the other four protein detected the OD value of the average, t-test difference has statistical significance, suggesting that objective strong antigenicity in the other four protein.3.p166,p179,pORF2aa439?660,pORF2aa422?637,pORF2aa430?6 60 immune serumHEV cell culture is very difficult, there is no conventional neutralization test in vitro, and no sensitive to small animal model. So this experiment using based on PCR HEV neutralization test in vitro and to evaluate the objective, namely, pORF2aa439?660, pORF2aa422?637, pORF2aa430?660 immune mouse serum neutralizing activity. The results are as follows:A. Objective, namely, pORF2aa439?660, pORF2aa422?637 and pORF2aa430?660 in mice immune sera had neutralizing effect, and the titer were 1:8,1:8,1:80,1:80 1:4.pORF2aa439?660, pORF2aa422?637 immune serum neutralization titers highest, pORF2aa430?660 immune serum neutralization effect price lowest.B. By bioinformatics analysis software and the objective, namely, pORF2aa439?660, pORF2aa422?637, prominent situation pORF2aa430?660 neutralizing epitope for the analysis. The results showed that the five protein neutralizing antigenic table an outstanding area were 12.93,12.01,9.681,11.24 and 9.372. The data illustrate five protein dimeric natural state under the objective to highlight the extent the most obvious, pORF2aa430?660 protrusion of the weakest can partly explain the objective antigenicity is better than the other four truncated proteins.Conclusion:1. On the basis of these results indicate that of N-terminal and C-terminal extension, can obtain contains more antigenic epitopes of bit, with good solubility, and dimer form of HEV ORF2 truncated proteins.2. A new truncated proteins pORF2aa439?660, pORF2aa422?637, objective and pORF2aa430?660, namely of comparison, it is found that the five protein showed good immunogenicity and antigenicity. PORF2aa439?660, pORF2aa422-637, immune pORF2aa430?660 raw is better than that of the objective, namely, and objective antigen is better than that of the pORF2aa439?660, pORF2aa422?637, pORF2aa430?660 and these results indicate that;Have neutralizing activity of immune serum. pORF2aa439?660, pORF2aa422?637, pORF2aa430-660 induced, but neutralizing titer immune serum pORF2aa430?660 fragments of the lowest, pORF2aa439-660, high titers of neutralizing antibody in the serum of immune pORF2aa422?637. The two truncated proteins have may as recombinant hepatitis E vaccine candidate fragment, it is worth in-depth study. |
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