Tuberculosis subunit vaccine containing unite adjuvant

A subunit vaccine and combined adjuvant technology, applied in the field of new subunit vaccines, can solve the problems of weak immunogenicity and poor response effect, and achieve the effects of enhancing cellular immunity, enhancing Th1 type response, and enhancing humoral immune response.

Active Publication Date: 2013-11-13
NAT INST FOR FOOD & DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the main disadvantage of subunit vaccines is weak immunogenicity, and effective adjuvant assistance is often required to elicit an ideal immune response.

Method used

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  • Tuberculosis subunit vaccine containing unite adjuvant
  • Tuberculosis subunit vaccine containing unite adjuvant
  • Tuberculosis subunit vaccine containing unite adjuvant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1Ag85

[0024] Example 1 Preparation of Ag85b and ESAT6-CFP10 antigens

[0025] 1.1 Test method

[0026] 1) Amplification of Ag85b gene and purification of protein

[0027] Query the nucleic acid and amino acid sequences of Mycobacterium tuberculosis H37Rv antigen ESAT6 and CFP10 from Genbank, and design primers according to the target sequence.

[0028] Table 1

[0029]

[0030] First, the H37Rv whole genome was used as a template, and the primers are shown in Table 1. Ag85b was amplified in a 50μL PCR system (ddH 2 O30.5μL, 10×Buffer 5μL, Taq enzyme 0.5μL, dNTP4μL, upstream primer 4μL, downstream primer 4μL, DNA template 2μL). The amplification conditions were: 94°C for 10 min; 94°C for 45s, 62.2°C for 45s, 72°C for 1 min, 33 cycles; 72°C for 5 min.

[0031] After agarose gel electrophoresis of the PCR product, the gel was recovered to obtain the target fragment, which was digested by NdeI and EcoRI and ligated with the pET30a cloning expression vector under the catalysis of T4 DNA ligase at ...

Embodiment 2

[0045] Example 2: Immunological study of the tuberculosis subunit vaccine containing the combined adjuvant in the present invention

[0046] 1. Material

[0047] Research objects: 30 SPF female BALB / c mice (6-8 weeks old)

[0048] 2. Methods and results

[0049] 2.1 Experimental design

[0050] Thirty female BALB / c mice were randomly divided into 5 groups, each with 6 mice. The mice were similar in age and body weight. The test groups are shown in Table 2: (EC is ESAT6-CFP10)

[0051] table 3

[0052] Group

Immunization dose ( / only)

Aluminum+Poly IC

Al(OH) 3 (0.2mg)+poly IC(50μg)

Protein

Ag85b(10μg)+EC(10μg)

Protein + Aluminum

Ag85b(10μg)+EC(10μg)+Al(OH) 3 (0.2mg)

[0053] Protein+PolyIC

Ag85b(10μg)+EC(10μg)+poly IC(50μg)

Protein + Aluminum + PolyIC

Ag85b(10μg)+EC(10μg)+Al(OH) 3 (0.2mg)+poly IC(50μg)

[0054] The mice were immunized with the above reagents intramuscularly on the hind legs of the mice, and immunized with 3 injections at 10 days intervals. Splenic...

Embodiment 3

[0058] Example 3 Animal protection test

[0059] 1. Experimental method

[0060] Immunotherapy of Mtb infection in guinea pigs

[0061] 16 SPF Hartley guinea pigs (300-350g / mouse), half male and half female, were divided into two groups (experimental group and control group), each with 8 animals. The experimental group and the control group guinea pigs were challenged subcutaneously 5.0×10 3 After CFU Mtb, the experimental group was treated with the reference vaccine, a total of 6 injections, with an interval of 2 weeks between each injection, the dose of each injection was [Ag85b(10μg)+EC(10μg)+Al(OH) 3 (0.2mg)+poly IC(50μg)] / 0.5mL / only. Guinea pigs in the control group were injected with the same amount of saline as a control. One week after the last immunization, the guinea pigs were dissected. Analyze the comprehensive pathological index of liver, spleen and lungs and the bacterial load of spleen and lungs.

[0062] Organ disease index score

[0063] After Mtb-infected guinea pi...

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Abstract

The invention provides a novel tuberculosis mycobacterium subunit vaccine containing unite adjuvant, which takes Ag85b protein, ESAT 6-CFP10 fusion protein as antigen component, and aluminum and PolyIC as a composite adjuvant. The adjuvant provided by the invention can effectively improve cellular immunity response of body to the tuberculosis subunit vaccine; at the same time, the adjuvant combines with the tuberculosis mycobacterium antigen Ag85b protein, ESAT6-CFP10 fusion protein, therefore the immunization effect is better than the compatibility effect of using other single adjuvant component and Ag85b protein and ESAT6-CFP10 fusion protein.

Description

Technical field [0001] The invention relates to a tuberculosis subunit vaccine, in particular to a novel subunit vaccine using Ag85b protein and ESAT6-CFP10 fusion protein as antigen components and aluminum and Poly IC as a composite adjuvant. Background technique [0002] Tuberculosis is an ancient and long infectious disease caused by Mycobacterium tuberculosis. In recent years, with the increase of multi-drug resistant strains of Mycobacterium tuberculosis and the continuous epidemic of AIDS, tuberculosis has revived. Tuberculosis has become a global public health problem. [0003] The World Health Organization released a report on the status of global tuberculosis in 2012. The report showed that in 2011 there were 8.7 million new tuberculosis cases and more than 1.4 million tuberculosis deaths worldwide. It can be seen from the report that the total number of TB cases, illnesses and deaths is still high, and the economic burden caused by it is very heavy. my country is the w...

Claims

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Application Information

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IPC IPC(8): A61K39/39A61K39/04A61P31/06
Inventor 王国治卢锦标都伟欣陈保文杨蕾苏城沈小兵
Owner NAT INST FOR FOOD & DRUG CONTROL
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