Recombinant lactobacillus and use of the same

A technology of Lactobacillus and Lactobacillus casei, which is applied in the direction of bacteria, drug combinations, and medical preparations containing active ingredients, etc., can solve the problems of unsatisfactory treatment of mite allergy and impossibility of disease prevention

Inactive Publication Date: 2009-07-01
NAT UNIV OF SINGAPORE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Therefore, current treatment of mite allergy is unsatisfactory and disease prevention is not possible; there is thus a long-felt need for improved and effective therapeutic and preventive strategies

Method used

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  • Recombinant lactobacillus and use of the same
  • Recombinant lactobacillus and use of the same
  • Recombinant lactobacillus and use of the same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0179] Example 1: Construction of recombinant Lactobacillus casei expressing enhanced green fluorescent protein (eGFP) using extension high-fidelity DNA polymerase (Boehringer) and synthetic primers BamHI-eGFP / f[5′-CCC CCG GAT CCA gtg agc aag ggc gag gag ctg-3', SEQ ID NO: 3] and eGFP-xhoSac / r[5'-CCC CCC ctc gag CTT GTA CAG CTC GTC CATGCC GAG-3', SEQ ID NO: 4] were analyzed by polymerase chain reaction ( PCR) to amplify the eGFP gene. The resulting PCR products (kits) containing a Bam HI site at the 5' end and an Xho I / Sac I site at the 3' end were then subcloned into pTUAT at Bam HI and Sac I (cf. figure 1 ). The Bam HI / Nhe I fragment containing eGFP-uidA-Tbch was exchanged with the Bam HI / Nhe I fragment expression-secretion vector of pLP500 (refer to figure 1 ).

[0180] The uidA gene was then removed by digestion with Xho I, resulting in the expression construct pLP500-eGFP.

Embodiment 2

[0181] Example 2: Cloning of Der p2 and Blo t5 genes into a Lactobacillus / E. coli shuttle vector

[0182] Using extension high-fidelity DNA polymerase (Boehringer) and synthetic primers, Dp2Bam / f [5'-CCCCCGGATCCAGATCAAGTCGATGTCAAAGATTGTGC-3', SEQ ID NO:5] and Dp2xhoSac / r [5'-CCCCCCGAGCTCCTCGAGATCGCGGATTTTAGCATGAGTAGC-3', SEQ ID NO:6] The 441 bp fragment of Der p2 cDNA was amplified by PCR. The resulting PCR product of the Der p2 gene containing Bam HI and Xho I / Sac I sites at the 5' and 3' ends, respectively, has the sequence: 5'-GGATCC A GAT CAA

[0183] GTC GAT GTC AAA GAT TGT GCC AAT CAT GAA ATC AAA AAA GTT TTG GTA

[0184] CCA GGA TGC CAT GGT TCA GAA CCA TGT ATC ATT CAT CGT GGT AAA CCA

[0185] TTC CAA TTG GAA GCC GTT TTC GAA GCC AAC CAA AAC ACA AAA ACC GCT

[0186] AAA ATT GAA ATC AAA GCC TCA ATC GAT GGT TTA GAA GTT GAT GTT CCC

[0187] CGT ATC GAT CCA AAT GCA TGC CAT TAC ATG AAA TGC CCA TTG GTT AAA

[0188] GGA CAA CAA TAT GAT ATT AAA TAT ACA TGG AAT GTT CCG AAA ATT...

Embodiment 3

[0203] Example 3: Confocal analysis of Lactobacillus casei Shirota-eGFP

[0204] Lactobacillus casei Shirota cells containing the pL500-eGFP construct (L. casei Shirota-eGFP) and the pL500 vector, respectively, were in MRS medium (Difco Laboratories Detroit) containing 5 μg / ml erythromycin at 37° C. 5.0% CO 2 Cultivated in an incubator. When the culture reaches an OD of 0.6 to 1.8 690nm , a total of 0.5 ml was collected and washed twice in PBS (pH 7.4). Cells were resuspended in 1 ml PBS and analyzed under a confocal microscope. Lactobacillus casei Shirota containing the pL500 vector was used as a negative control.

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Abstract

The invention relates to recombinant lactobacillus, use of the same and a pharmaceutical composition including the same. The recombinant lactobacillus includes a heterologous nucleic acid sequence. The heterologous nucleic acid sequence encodes at least an immunogenic fragment of the mite allergens Der p 1, Der p 2, or Blo t 5, or an immunogenic homolog thereof. A respective fragment of Der p 1 includes at least 8 % of the amino acid sequence of the mite allergen. A method of modulating the immune response to an allergen in a mammal as well as a pharmaceutical composition and a kit are also disclosed.

Description

technical field [0001] The present invention relates to recombinant lactobacillus and uses thereof. The recombinant Lactobacillus comprises a heterologous nucleic acid sequence encoding at least an immunogenic fragment of a mite allergen or an immunogenic homolog thereof. The present invention also relates to methods, pharmaceutical compositions and kits for modulating the immune response of a mammal to an allergen. Background technique [0002] Allergic diseases are thought to affect 25% to 40% of the population in developed countries, with more than half of Americans allergic to one or more allergens. In the UK, the rate of allergic reactions is increasing rapidly (especially in children) at a rate of about 5% per year. Allergies such as allergic rhinitis (hay fever), asthma and urticaria are hypersensitivity of the immune system response (ie the pathological response of the immune system to each allergen). In susceptible individuals, generally innocuous substances such...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21A61K39/00
CPCA61K2039/523A61K2039/521C07K14/43531A61K39/35A61K2039/542A61P37/00A61P37/08
Inventor 蔡考圆林丽芳陈丽娟
Owner NAT UNIV OF SINGAPORE
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