A kind of vaccine for preventing tuberculosis and combination drug and preparation method and application
A technology for tuberculosis and vaccines, which is applied in the combined drug and application field for preventing tuberculosis, can solve the problems of many types of adjuvants, the existence of hypersensitivity and Koch reaction risks, and poor efficacy, so as to reduce the treatment cycle, reduce the risk of Koch reaction, Effect of Absolute Usage Reduction
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Embodiment 1
[0073] Preparation of Tuberculosis Vaccine Containing Mycobacterial Microorganism Compound Adjuvant
[0074] 1. Preparation process of Ag85b protein
[0075] Find the gene sequence of Ag85b protein from GeneBank, and design primers as follows: see Table 1.
[0076] Table 1 Primer sequences, restriction sites and protected bases of Ag85b target gene
[0077]
[0078] Remarks: The underline is the restriction site of the endonuclease, and the italic part is the start codon.
[0079] The Ag85b gene was amplified by PCR using the whole genome of Mycobacterium tuberculosis H37Rv as a template. The PCR reaction system was 50 μL (ddH 2 O 30 μL, 10×Buffer 5 μL, dNTP 4 μL, P1 primer 4 μL, P2 primer 4 μL, DNA template 2.5 μL, DNA polymerase 0.5 μL), the amplification conditions are: 96 ℃ pre-denaturation for 3 minutes; 96 ℃ denaturation for 45 seconds, 62 Annealing at °C for 45 seconds, extension at 72 °C for 1 minute, a total of 30 cycles; extension at 72 °C for 7 minutes.
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Embodiment 2
[0109] Animal Protection Test of Tuberculosis Vaccine Containing Mycobacterial Microorganism Compound Adjuvant
[0110] 1. Experimental method
[0111] Immunotherapy of Mtb-infected guinea pigs
[0112] Sixteen SPF Hartley guinea pigs (300-350 g / pig), half male and half male, were divided into two groups (vaccine group and control group), with 8 animals in each group. The guinea pigs of the vaccine group and the control group were subcutaneously challenged with 5.0×10 3 CFU Mtb, 4 weeks later, the vaccine group was injected with this vaccine for treatment, a total of 6 injections, each injection was 1 week apart, and the dose of each injection was [Ag85b(10μg)+EC(10μg)+MV(22.5μg)] / 0.5mL / Only. The guinea pigs in the control group were injected with the same amount of normal saline as a control. After 12 weeks of Mtb infection, guinea pigs were dissected. The comprehensive lesion index of liver, spleen and lung and the bacterial load in spleen and lung were analyzed. Mtb ...
Embodiment 3
[0124] Comparative test of animal protection between tuberculosis vaccine containing mycobacterial microbial compound adjuvant and subunit tuberculosis vaccine
[0125] 1. Experimental method
[0126] Immunotherapy of Mtb-infected guinea pigs
[0127] Sixteen SPF Hartley guinea pigs (300-350 g / pig), half male and half male, were divided into two groups (vaccine group and control group), with 8 animals in each group. The guinea pigs of the vaccine group and the control group were subcutaneously challenged with 5.0×10 3 CFU Mtb, 4 weeks later, the vaccine group was injected with this vaccine for treatment, a total of 6 injections, each injection was 1 week apart, and the dose of each injection was [Ag85b(10μg)+EC(10μg)+MV(22.5μg)] / 0.5mL / Only. Guinea pigs in the control group were injected with the same amount of subunit tuberculosis vaccine ([Ag85b(10μg)+EC(10μg)+BCG-CpG-DNA(75μg)+Al(OH) 3 (200 μg)] / 0.5 mL / piece) as a control. After 12 weeks of Mtb infection, guinea pigs wer...
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