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A kind of non-resistance screening kiss1 eukaryotic expression plasmid and its application

A technology for expressing plasmids and resistance screening, which is applied in the direction of antibacterial drugs, bacterial antigen components, and the use of vectors to introduce foreign genetic materials. It can solve problems such as increased costs, unfavorable vaccine promotion and application, and trace allergy. High-impact, easy-to-save effects

Active Publication Date: 2017-11-24
HUAZHONG AGRI UNIV
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

In the process of cultivating bacteria, it is necessary to add such resistant substances to screen recombinant bacteria, which not only increases the cost, but also may lead to the dissemination of clinical drug-resistant strains and potential trace allergy in the process of gene vaccine product preparation (Han YG, etc. Efficacy and safety of an oral somatostatin DNA vaccine without antibiotic resistance gene in promoting growth of piglets.Scand J Immunol,2014,79(4):244-50), which is not conducive to the promotion and application of vaccines

Method used

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  • A kind of non-resistance screening kiss1 eukaryotic expression plasmid and its application
  • A kind of non-resistance screening kiss1 eukaryotic expression plasmid and its application
  • A kind of non-resistance screening kiss1 eukaryotic expression plasmid and its application

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Embodiment 1

[0018] Embodiment 1: Construction and large-scale preparation of non-resistance screening KISS1 eukaryotic expression plasmid pVAX-KISS1-S-asd

[0019] First synthesize the human KISS1 gene, which contains a HindIII restriction site at the 5' end and an EcoRI restriction site at the 3' end, and clones it into the HindIII and EcoRI restriction enzyme restriction sites of the eukaryotic expression vector pVAX1 (purchased from Invitrogen). site, the plasmid pVAX-KISS1 was obtained; then the plasmid pCMV-S was used as a template to amplify the HBsAg S gene, and then cloned into the NheI and HindIII restriction sites of the plasmid pVAX-KISS1 to obtain the intermediate plasmid pVAX - KISS1-S; replace the kanamycin gene (Kan) in the intermediate plasmid pVAX-KISS1-S with a non-resistance selection gene, the aspartate β-galactose dehydrogenase gene (asd), and will replace The resulting plasmid was transformed into Escherichia coli lacking the asd gene, and the positive clone x6097 / pV...

Embodiment 2

[0079] Embodiment 2: the application of animal immune castration gene vaccine of the present invention on sheep

[0080] 2.1 Vaccine immunization and sample collection

[0081] Six 8-week-old Hu sheep male lambs were randomly divided into 2 groups (purchased from Hubei Hengtai Sheep Breeding Co., Ltd.), with 3 lambs in each group. 24 hours before immunization, the animals were pretreated with 4 ml of 0.25% procaine hydrochloride intramuscularly injected into the quadriceps muscle at three points. Group T is the vaccine injection group, and the pVAX-KISS1-S-asd plasmid is injected intramuscularly at a dose of 1 mg per animal. The specific operation is to dissolve 1 mg of the plasmid vaccine in 4 ml of normal saline and then inject it at three points in the quadriceps of Hu sheep. C The control group was an empty plasmid control group, and the naked plasmid pVAX-asd was injected intramuscularly at a dose of 1 mg per mouse, and the specific operation was the same as that of the ...

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Abstract

The invention belongs to the field of gene vaccines, and discloses a non-antibiotic screening KISS1 eukaryotic expression plasmid pVAX-KISS1-S-asd. The nucleotide sequence of the non-antibiotic screening KISS1 eukaryotic expression plasmid pVAX-KISS1-S-asd is shown as SEQ ID NO:1. The invention further discloses a recombined escherichia coli x6097 / pVAX-KISS1-S-asd containing the non-antibiotic screening KISS1 eukaryotic expression plasmid pVAX-KISS1-S-asd; the recombined escherichia coli x6097 / pVAX-KISS1-S-asd is preserved in the China Center for Type Culture Collection; and the preservation serial number is CCTCC NO: M2014503. A bacterial strain can be used for extracting a large number of plasmids pVAX-KISS1-S-asd, and an animal immune castration gene vaccine is prepared. A recombined vaccine can be used for Hu sheep male lamb immune; immune responses can be effectively induced; the gonad function and the sexual behavior are restrained; and the aim of immune castration is achieved. The non-antibiotic screening immune castration gene vaccine is low in cost, obvious in effect, convenient to use, safe, reliable, easy to store and capable of being used for immune castration of various mammals.

Description

technical field [0001] The invention belongs to the field of gene vaccines. It specifically relates to a non-resistance screening KISS1 eukaryotic expression plasmid, recombinant Escherichia coli containing the eukaryotic expression plasmid, and the application of the recombinant Escherichia coli in preparing animal immune castration gene vaccines. The present invention also relates to an animal immune Preparation method of castration gene vaccine. Background technique [0002] The castration of domestic animals has a long history in our country. It was first recorded in "Xia Xiaozheng" of the Xia Dynasty, and the castration of male animals was called Gongju or Gongte. At present, the method of castration of livestock is still mainly surgical castration. China's annual slaughter of livestock is huge, including 600 million pigs and 300 million sheep (Mi Chungai. National Animal Husbandry Development Overview. Yearbook of the People's Republic of China, 2012). In animal hus...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/79C12N1/21A61K39/07A61P31/04
Inventor 姜勋平韩燕国刘桂琼
Owner HUAZHONG AGRI UNIV
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