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Human replication-deficient recombinant adenovirus vector for efficiently expressing leishmania PEPCK

A technology of recombinant adenovirus and Leishmania, which is applied to viruses/phages, medical preparations containing active ingredients, viruses, etc., can solve the problems of highly weakened pathogenicity and impaired proliferation of Leishmania, and achieve High safety effect

Inactive Publication Date: 2021-09-03
SHANXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Targeted loss of PEPCK leads to impaired proliferation and highly attenuated pathogenicity in Leishmania

Method used

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  • Human replication-deficient recombinant adenovirus vector for efficiently expressing leishmania PEPCK
  • Human replication-deficient recombinant adenovirus vector for efficiently expressing leishmania PEPCK
  • Human replication-deficient recombinant adenovirus vector for efficiently expressing leishmania PEPCK

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Preparation of recombinant adenovirus vector Ad5-L.m-PEPCK:

[0039] 1. According to the coding sequence of Leishmania PEPCK gene (NCBI GenBank database entry XM_003721939.1; Leishmania major strain Friedlin) (a total of 1575 nucleotides, nucleotides, encoding 525 amino acids), the carboxyl terminal containing 6x Histidine (6xHis) was synthesized The full-length PEPCK gene. Add a sequence containing a NotI restriction site in front of the start codon ATG GCGGCCGC ACC; add the sequence encoding 6x His before the stop codon TGA, and add the sequence of EcoRV restriction enzyme cutting site behind GATATC ( figure 1 ).

[0040] 2. Add 11 additional modified amino acids GRALEHHHHHH to the carboxyl terminus of the PEPCK gene containing a 6x His tag at the carboxyl terminus, so that the expressed protein contains a total of 536 amino acids.

[0041] 3. By using NotI and EcoRV double enzyme digestion, the above-mentioned modified PEPCK gene and the shuttle vector pShuttle...

Embodiment 2

[0048] Ad5-L.m-PEPCK separation and purification:

[0049] In order to obtain a large amount of purified recombinant adenovirus vectors, 293 cells were infected with the primary vector Ad5-L.m-PEPCK in Example 1, and the multiplicity of infection (MOI) was 1 plaque forming unit (PFU). 48 hours after infection, infected cells were harvested and freeze-thawed 3 times. Cell lysates were subjected to stepwise gradient centrifugation with 1.2 and 1.4 g / ml CsCl and ultracentrifuged at 35,000 rpm for 2 h. Harvesting the main strip ( image 3 The position indicated by the middle arrow), and then dialyzed into the dialysate (100mM sucrose; 10mM Tris-HCl, pH 8.0; 2mM MgCl 2 ), stored at -80°C. Dialysis At 4°C, dialyze for 24 hours, and change the dialysate every 6 hours, 2-3 liters of dialysate each time.

Embodiment 3

[0051] Purification and immunoassay of Ad5-L.m-PEPCK protein expressed in cells (PEPCK):

[0052] 293 cells were infected with Ad5-L.m-PEPCK preserved in Example 2, and the multiplicity of infection (MOI) was 1 plaque forming unit (PFU). After 48 hours of infection, the infected cells were harvested and washed with 4°C phosphate-buffered saline (PBS, 137mM NaCl; 2.7mM KCl; 10mM NaCl 2 HPO 4 ; 1.8mM KH 2 PO 4 ) wash, in containing 50mM NaH 2 PO 4 buffer [pH 7.4, 300 mM NaCl, 10 mM imidazole, 0.5% Triton X-100, 10% glycerol and protease inhibitor cocktail (Roche)], and then centrifuged at 22,000×g for 30 min at 4°C. Ni-nitrilotriacetic acid (NTA) agarose (Qiagen) was added to the clarified supernatant and incubated at 4°C for 2 hours to capture His-tagged proteins. The precipitated protein was eluted with 250 mM imidazole solution, separated on 10% polyacrylamide gel electrophoresis (SDS-PAGE), and stained with Coomassie brilliant blue R250 ( Figure 4 ) and Western blot ...

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Abstract

The invention belongs to the field of biotechnology, and discloses a human replication-deficient recombinant adenovirus vector for efficiently expressing leishmania phosphoenolpyruvate carboxykinase (PEPCK) aiming at the current situation of leishmania. Specifically, carrying out homologous recombination on a recombinant vector pShuttle-CMV-Leish-PEPCK constructed by a modified PEPCK gene and a pShuttle-CMV vector and a pAdEasy-1 vector, carrying out PacI cutting on the obtained plasmid vector pAd5-L.m-PEPCK, and transfecting into a human embryo kidney cell 293, so as to obtain the replication-deficient recombinant adenovirus vector Ad5-L.m-PEPCK. Through Coomassie brilliant blue R250 dyeing and Western blot analysis of a specific anti-His tag antibody, it is shown that the recombinant adenovirus vector Ad5-L.m-PEPCK can efficiently express the PEPCK protein. As the leishmania phosphoenolpyruvate carboxykinase (PEPCK) is the most immunodominant leishmania disease antigen which is found recently. Therefore, the recombinant adenovirus vector Ad5-L.m-PEPCK disclosed by the invention can be used for a vaccine for the leishmaniasis, so that the prevention and control of the leishmaniasis are facilitated.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a human replication-deficient recombinant adenovirus vector for highly expressing Leishmania PEPCK. Background technique [0002] Leishmaniasis (leishmaniasis), also known as kala-azar. Its causative agents are several viscerotropic Leishmania species. The protozoan mainly parasitizes in the macrophages in the patient's body, and is transmitted by the dipteran insect sandfly. The main clinical features are long-term irregular fever, splenomegaly, anemia, emaciation, leukopenia and increase of serum globulin. If proper treatment is not given, most of the patients die within 1-2 years after getting the disease due to complications from other diseases. [0003] The World Health Organization has listed kala-azar as a parasitic disease that is on the rise again. The distribution of kala-azar is extremely wide, affecting the four continents of Asia, Europe, Africa and Latin America. K...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/861C12N15/30A61K39/008A61P33/02
CPCC12N15/86C07K14/44A61K39/0008A61P33/02C12N2710/10043Y02A50/30
Inventor 邢力吴长新潘元晴郭凡
Owner SHANXI UNIV
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