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Novel genetic engineering subunit vaccine for chicken mycoplasma synoviae

A technology of Mycoplasma synovialis and amino acids, which is applied in genetic engineering, vaccines, veterinary vaccines, etc., can solve the problems of difficult mycoplasma culture, low antigen content, weak autoimmune protection, etc., and achieves good immunogenicity and immunogenicity. The effect of strong performance and low production cost

Inactive Publication Date: 2019-07-19
苏州世诺生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The vaccines currently used to prevent and control Mycoplasma synovialis infection are all traditional inactivated vaccines, but the cultivation of Mycoplasma is difficult, serum is required, the antigen content is low, and the inactivated vaccine has the problem of weak autoimmune protection

Method used

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  • Novel genetic engineering subunit vaccine for chicken mycoplasma synoviae
  • Novel genetic engineering subunit vaccine for chicken mycoplasma synoviae
  • Novel genetic engineering subunit vaccine for chicken mycoplasma synoviae

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0113] Example 1 Construction and Identification of Transfer Vector pF-MSPA

[0114] 1. MSPA gene amplification and purification The codon-optimized MSPA gene (SEQ ID NO: 1) was synthesized in Nanjing GenScript Company and cloned into the pUC17 vector to obtain the pUC-MSPA plasmid vector. The pUC-MSPA plasmid was used as a template, and MSPA-F and MSPA-R were used as upstream and downstream primers for PCR amplification (the gene sequences of MSPA-F and MSPA-R are shown in SEQ ID NO.5 and 6). For the amplification system, see Table 1.

[0115] Table 1 MSPA gene amplification system

[0116]

[0117] The reaction conditions were: 95°C pre-denaturation for 5 minutes; 94°C denaturation for 45 seconds, 54°C annealing for 45 seconds, 72°C extension for 1 minute, 35 cycles; 72°C extension for 10 minutes.

[0118] Perform gel electrophoresis on the PCR product to verify the size of the target gene, such as figure 1 As shown, the target band appeared at the position of 1.3kbp, ...

Embodiment 2

[0130] Example 2 Construction and Identification of Transfer Vector pF-MSPB

[0131] 1. MSPB gene amplification and purification The codon-optimized MSPB gene (SEQ ID NO: 3) was synthesized in Nanjing GenScript and cloned into the pUC17 vector to obtain the pUC-MSPB plasmid vector. The pUC-MSPB plasmid was used as a template, and MSPB-F and MSPB-R were used as upstream and downstream primers for PCR amplification (the gene sequences of MSPB-F and MSPB-R are shown in SEQ ID NO.7 and 8), and the amplification system is shown in table 5.

[0132] Table 5 MSPB gene amplification system

[0133]

[0134] The reaction conditions were: 95°C pre-denaturation for 5 minutes; 94°C denaturation for 45 seconds, 54°C annealing for 45 seconds, 72°C extension for 1 minute, 35 cycles; 72°C extension for 10 minutes.

[0135] Perform gel electrophoresis on the PCR product to verify the size of the target gene, such as image 3 As shown, the target band appears at the position near 1.0kbp, ...

Embodiment 3

[0147] Example 3 Construction of recombinant baculovirus genome Bac-MSPA and Bac-MSPB

[0148] 1. Transformation of DH10Bac bacteria Take 1 μl of pF-MSPA plasmid in Example 1 and 1 μl of pF-MSPB plasmid in Example 2 and add it to 100 μl of DH10Bac competent cells, mix evenly, ice-bath for 30 minutes, and heat shock in 42°C water bath for 90 seconds. Cool on ice for another 2 minutes, add 900 μl LB liquid medium without Amp, and incubate at 37° C. for 5 hours. After 100 μl of bacterial solution was diluted 81 times, 100 μl of diluted bacterial solution was spread on LB solid medium containing gentamicin, kanamycin, tetracycline, X-gal and IPTG, and cultured at 37°C for 48 hours.

[0149] 2. Select single clones and use inoculation needles to pick up large white colonies, then streak on LB solid medium containing gentamicin, kanamycin, tetracycline, X-gal and IPTG, and culture at 37°C for 48 hours , and then pick a single colony to inoculate the LB liquid medium containing gent...

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Abstract

The application of the invention provides an immune composition and a subunit vaccine. The immune composition contains chicken mycoplasma synoviae MSPA (Mycoplasma synoviae surface protein A) proteincoded with a nucleic acid molecule of which the nucleotide sequence is as shown in SEQID NO:1 or a nucleic acid molecule of which the nucleotide sequence is 95% or above the same as that as shown in SEQID NO:1, and mycoplasma synoviae MSPB (Mycoplasma synoviae surface protein B) protein coded with a nucleic acid molecule of which the nucleotide sequence is as shown in SEQID NO:3 or a nucleic acidmolecule of which the nucleotide sequence is 95% or above the same as that as shown in SEQID NO:3. Sf9 cells of the vaccine are used for expressing MSPB protein and MSPA protein, the antigenicity, theimmunogenicity and the function of the product namely the immune composition are similar to those of natural protein, and the immune composition is high in expression level and high in immunogenicity, does not have pathogenicity to chickens; and besides, the vaccine can be prepared through large-scale serum-free suspension culture with a bioreactor, and besides, the production cost of the vaccinecan be greatly reduced.

Description

technical field [0001] The application relates to the technical field of animal immunity medicines, in particular to a novel genetically engineered subunit vaccine of Mycoplasma gallinarum. Background technique [0002] Chicken synovial mycoplasma disease, also known as chicken infectious synovitis, is an infectious disease of young chickens and turkeys caused by Mycoplasmasynoviae (MS), also known as chicken infectious synovitis (avian nonfectious synovitis) ), the disease mainly involves the joint synovial fluid and tendon sheath, which is characterized by swelling of joints, tendon sheaths and soles. Infection of flocks with the disease can result in significant lameness, stunted growth and carcass degradation. The disease mainly affects commercial broilers, laying hens and breeders, and can occur throughout the year. [0003] Mycoplasma gallinarum was a polymorphic spheroid with a negative Gram stain. There is only one serotype, and the pathogenicity of different stra...

Claims

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Application Information

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IPC IPC(8): A61K39/02A61P31/04C12N15/31
CPCA61K39/0241A61K2039/523A61K2039/552A61K2039/70A61P31/04C07K14/30
Inventor 曹文龙孔迪滕小锘易小萍张大鹤
Owner 苏州世诺生物技术有限公司
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