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Expression of a single chain antibody against salmonella in lactobacillus

一种单链抗体、沙门氏菌的技术，应用在微生物学和基因工程领域，能够解决血清变型等问题

Active Publication Date: 2017-08-18
萨姆巴德哈高什
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, effective vaccines have been developed against some host-adapted and common serovars (in host primary sources), but their use has led to the emergence of other serovars

Method used

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  • Expression of a single chain antibody against salmonella in lactobacillus
  • Expression of a single chain antibody against salmonella in lactobacillus
  • Expression of a single chain antibody against salmonella in lactobacillus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0411] Isolation and Characterization of Lactobacilli

[0412] Various organs (trachea, crop, gizzard, small intestine, large intestine and ceacum) were collected from backyard poultry birds. Lactobacilli were isolated by inoculating field samples in Lactobacillus selective liquid medium (LSB) (HiMedia / M1166-500G) and incubating at 37°C under anaerobic conditions. Selected colonies were enriched on MRS liquid medium (HiMedia / M369-500G). The purity of the selected colonies was checked by Gram stain (Gram positive short rods). The genetic identity of Lactobacillus was confirmed by performing a sequencing reaction of 900 bp amplicons as shown in SEQ ID NO: 245 (forward primer) and SEQ ID NO: 246 (reverse primer) The primers amplify the 16S RNA gene. The genetic identity of L. reuteri was confirmed by performing a sequencing reaction of the 303bp amplicon represented by SEQ ID NO: 247 (forward primer) and SEQ ID NO: 248 (reverse primer). The indicated species-specific primers ...

Embodiment 2

[0414] Isolation of Lactobacillus reuteri MuB and CnBP

[0415] Genomic DNA was isolated from the 1LB7 strain by resuspending the bacterial pellet in 5 ml of TNE buffer containing lysozyme (10 mg / ml concentration). 500 μl of 10% SDS and 250 μl of proteinase K (concentration of 10 mg / ml) were added and incubated at 55° C. for 2 hours with intermittent shaking. Genomic DNA was isolated using the phenol chloroform extraction method (Raya et al., Food Microbiology Protocols, 2001, 14, 135-139).

[0416] A partial 1.7 kb region of the MuB gene was PCR amplified using primers as shown in SEQ ID NO: 249 (forward primer) and SEQ ID NO: 250 (reverse primer). This 1.7 kb region contains the LPTQG motif. Amplicons were then cloned into pJet vectors and sequenced according to the manufacturer's instructions (Catalogue #: K1231, Thermo Scientific).

[0417] The entire CnBP gene (1.08 kb) was PCR amplified using primers as shown in SEQ ID NO: 251 (forward primer) and SEQ ID NO: 252 (reve...

Embodiment 3

[0419] Generation of camelid antibodies against whole-cell Salmonella

[0420] Immunization of camels with whole-cell killed Salmonella enterica: Briefly, actively growing Salmonella enterica cultures (log phase) were inactivated at 37°C for 24 hours by the addition of 0.5% formalin. The culture was maintained under constant shaking at 20 rpm. Cultures were then stored at 4°C and representative samples were tested for innocuity in enriched growth medium. After confirmation of inactivation, the bacterial culture was washed 3 times in 1X PBS buffer and resuspended at a concentration of 200 μg / ml. 5 ml of the suspension were mixed with an adjuvant (Montanide ISA 206V) to form an emulsion.

[0421] A final bleed of the immunized camels was performed 60 days after immunization. Total RNA was isolated from isolated peripheral blood lymphocytes. Use the primers shown in SEQ ID NO:253 (forward primer) and SEQ ID NO:254 (reverse primer) to carry out the PCR reaction that is used ...

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Abstract

The present disclosure relates to camel id antibodies that inhibit growth, and colonization of Salmonella serovars. The present disclosure also relates to a modified Lactobacillus as a delivery vehicle for controlling salmonella in a host organism.

Description

technical field [0001] This disclosure relates to the fields of microbiology and genetic engineering. The present disclosure provides recombinant chimeric proteins and antibodies against salmonella. [0002] Background of the invention [0003] Humans have developed and used fermented foods with the aid of lactic acid bacteria (LAB), which have been classified as probiotics and classified as recognized by the U.S. Food and Drug Administration (USFDA) as Safe (GRAS). In addition to being considered as powerhouses in the food industry, LABs continue to be the focus of considerable interest as probiotic organisms, as they have also been endowed with specific health-promoting functions, which they perform by modulating the intestinal environment of the host . [0004] Their ability to attach to certain regions of the gastrointestinal tract has sparked interest in the potential of such microorganisms as vehicles for the delivery of bioactive compounds and vaccines (Pouwels et a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/12C12N1/21C07K19/00C12N1/20C12R1/225
CPCC07K14/335C07K16/1235C07K2319/00C07K2317/73C07K2317/76C07K2317/569C07K2317/22C07K2317/14C12R2001/225C12N1/205C07K16/005C07K2317/622Y02A50/30A23K20/10A23L33/17A23L3/00A23V2002/00C07K2317/565
Inventor 萨姆巴德哈·高什萨基本·库马尔·班尔吉玛尼莎·普拉文·萨博尼斯
Owner 萨姆巴德哈高什
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