Hepatitis E-foot-and-mouth disease combined vaccine and preparation method thereof
A combined vaccine and hepatitis E technology, which is applied in the field of hepatitis E-foot-and-mouth disease combined vaccine and its preparation, can solve problems that have not been reported yet, and achieve lower vaccine management costs, lower production costs and market prices, good safety and Immunogenic effects
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Embodiment 1
[0036]A component of hepatitis E-foot-and-mouth disease (HEV-FMDV) combined vaccine, including foot-and-mouth disease inactivated vaccine and hepatitis E virus recombinant protein. Among them, the porcine foot-and-mouth disease inactivated vaccine has a virus antigen content of 0.5-5 μg / ml, and a hepatitis E virus recombinant protein content of 5-100 μg / ml. The virus in the porcine foot-and-mouth disease inactivated vaccine used in this example is derived from O / Mya98 / XJ / 2010+O / GX / 09-7 vaccine strain or other available vaccine strains. The amino terminal of the hepatitis E virus recombinant protein is located between the 380th and 480th amino acids of its ORF2 encoded protein, and its carboxyl terminal is located between the 580th and 660th amino acids or derivatives produced by this protein fragment, and its described sequence for (295). The amino acid sequence SEQ ID NO.1 is preferably used.
[0037] In this embodiment, the HEV-FMDV combined vaccine also contains an immune...
Embodiment 2
[0046] Expression and Purification of Hepatitis E Virus Recombinant Protein
[0047] 1. Using the gene sequence of type 4 HEV Chinese strain as a template, use primer 1 (5'-CCCCCATGGTTATCCAGGACTATGATAATC-3') and primer 2 (5'-CCCCTCGAGATACTCCCGGGTTTTTACCCC-3') to amplify the SEQ ID NO.2 gene fragment in ORF2. The PCR product was recovered and purified by agarose gel, and cloned into the plasmid vector Pet28(a)+.
[0048] 2. Transform Escherichia coli strain EL-21(DE3) with the plasmid carrying the target fragment, pick a single colony, and culture it to OD with LB medium containing 50ug / ml kanamycin 550 After reaching 0.6-0.8, add IPTG with a final concentration of 0.2-1.0mM for induction, the induction temperature is 37°C, the shaking speed is 200rpm, and the bacteria are collected by centrifugation after 3-4 hours, and the cell lysate (50mM Tris-HCL , pH7.2, 300mM NaCl) suspended cells, freeze-thawed 6 times, ultrasonically disrupted the cells, collected the supernatant by c...
Embodiment 3
[0050] Preparation method of porcine foot-and-mouth disease inactivated vaccine and hepatitis E virus recombinant protein ISA 206 gel adsorption solution
[0051] In order to increase the immunogenicity, the virus stock solution of porcine foot-and-mouth disease inactivated vaccine and the recombinant protein of hepatitis E were adsorbed on ISA 206 gel:
[0052] 1. Stir and mix the virus stock solution of porcine foot-and-mouth disease inactivated vaccine and ISA 206 gel;
[0053] 2. Stir and mix the two hepatitis E recombinant proteins with ISA 206 gel respectively.
[0054] 3. Mix the two mixed solutions of the above-mentioned gains in a certain proportion, adjust the virus antigen content of the porcine foot-and-mouth disease inactivated vaccine to be 2 μg / ml, the hepatitis E recombinant protein concentration to be 50 μg / ml, and the concentration of ISA 206 gel to be 50w / ml w%; the resulting mixed solution is the inactivated porcine foot-and-mouth disease vaccine and hepat...
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