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Transfection system for expressing exogenous gene in vagina trichmonad body

A technology of Trichomonas vaginalis and exogenous gene, applied in the field of transfection system, can solve problems such as lack of transfection system, and achieve the effect of easy purification and small viral genome

Inactive Publication Date: 2011-04-06
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Trichomonas vaginalis is a flagellate that parasitizes the urogenital tract. It not only causes vaginitis in humans, but also causes clinical symptoms in primate squirrel monkeys, and makes other animals such as mice become carriers. The disease is widely distributed. , the infection rate is high, and the harm is serious, but there is no ideal control method so far. The reason for this is the lack of a suitable transfection system for exogenous gene expression in Trichomonas vaginalis

Method used

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  • Transfection system for expressing exogenous gene in vagina trichmonad body
  • Transfection system for expressing exogenous gene in vagina trichmonad body
  • Transfection system for expressing exogenous gene in vagina trichmonad body

Examples

Experimental program
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Effect test

Embodiment 1

[0020] DNA-based transfection system

[0021] As shown in Figure 1, at first clone Trichomonas vaginalis a-SCSB (a-succinyl-CoA synthetase promoter) gene and carry out full sequence analysis; Apply PCR technology to amplify and clone Trichomonas vaginalis a-SCSB gene The promoter sequence 5SCSB and the poly(A) signal SCSB3 and GFP were introduced into the multiple cloning restriction site (MCS), and the green fluorescent protein (EGFP) was inserted between the gene promoters SCSB5 and SCSB3 by using the MCS, and cloned into Vector, construct the GFP expression cassette pa-SCSB5 / EGFP / a-SCSB3, and introduce the trophozoite of Trichomonas vaginalis by electroporation, the expression of GFP was detected by fluorescence microscope after 6 hours, and its transcriptional activity was determined; the same method was used to construct the NEO resistance cassette pa -SCSB5 / NEO / a-SCSB3 and transfected Trichomonas vaginalis, G418-resistant strains were obtained by G418 screening; GFP expr...

Embodiment 2

[0023] Transfection system using Trichomonas vaginalis virus RNA as carrier

[0024] As shown in Figure 2, the T7 promoter sequence was directly added when designing the PCR primers, and the T7 promoter was introduced into the front of the 5′ end of the full-length cDNA of Trichomonas vaginalis virus by PCR reaction, and then amplified by PCR. The 5' end 289bp (only including the 5' end UTRs) and the 3' end 174bp of TVV and the 720bp EGFP coding region three fragments were added, and the connection with the pMD18-T cloning vector was carried out. Kpn I double enzyme digestion, recover the target fragment of 289bp at the 5' end of TVV; use Kpn I / Xba I double enzyme digestion to recombined plasmid pEGFP, recover 720bp EGFP part; recombined plasmid pTVVS with Xba I / Pst I double enzyme Cut and recover the 174bp fragment at the 3' end of TVV; the vector pPoyII / sfinot was double digested with EcoR I / PstI. The obtained target fragment and the carrier were mixed and ligated in a mola...

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Abstract

The invention provides a transfection system of foreign gene which expresses in the body of a trichomonas vaginalis, which is a transfection system which utilizes trichomonas vaginalis virus to be transfection carrier and utilizes DNA to be transfection carrier. The invention comprises a transfection system which adopts DNA to be carrier and a transfection system which adopts trichomonas vaginalis virus RNA to be carrier. Trichomonas vaginalis virus has little genome, which is stable and is easy to be purified. Trichomonas vaginalis is a eukaryote, and protein which is expressed out by foreign gene in trichomonas vaginalis has activity. Trichomonas vaginalis is easy to culture in vitro, and exogenote can be prepared in large scale in vitro without limination as serial subcultivation.The transfection system contains polyclonal sites, which is universal transfection carrier, various foreign genes can express in the body of trichomonas vaginalis, which provide a transfection system for researching trichomonas vaginalis molecular biology and cell biology.

Description

Technical field: [0001] The invention provides a transfection system for exogenous gene expression in Trichomonas vaginalis, belonging to the field of genetic engineering. Background technique: [0002] Trichomonas vaginalis is a flagellate that parasitizes the urogenital tract. It not only causes vaginitis in humans, but also causes clinical symptoms in primate squirrel monkeys, and makes other animals such as mice become carriers. The disease is widely distributed. , the infection rate is high, and the harm is serious, but there is no ideal control method so far. The reason is the lack of a suitable transfection system for the expression of foreign genes in Trichomonas vaginalis. Invention content: [0003] The invention provides a transfection system for exogenous gene expression in Trichomonas vaginalis, which uses Trichomonas vaginalis virus as a transfection carrier and DNA as a transfection carrier. [0004] The transfection system contains polyclonal enzyme cuttin...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/09C12N15/79C12N1/10
Inventor 张西臣李建华张昕鑫宫鹏涛杨举
Owner JILIN UNIV
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