CN1325958A - Process for preparing genetically engineered recombinant human lysozyme - Google Patents

Abstract

A process for preparing genetically engineered recombinant human lysozyme is based on molecular biology and genetic enginering, and includes such steps as cloning human lysozyme gene to yeast body, expressing it, extracting and purifying. Its advantages include preparing human lysozyme in large batches and high stability and purity.

Description

A kind of method of producer gene engineered recombinant human lysozyme

The invention belongs to molecular biosciences pharmaceutical technology field, is a kind of method of producing human lysozyme with the genetically engineered recombination method.

At present, human lysozyme mainly extracts from people's milk and placenta tissue and obtains, and its source is limited, extracts complexity, and output is very low, is subject to human disease's pathogeny fungi pollution, uses so be mainly used in scientific research, can't supply clinical application.The lysozyme of chicken that from egg, extracts in addition, the output height, the source is easy, but because foreign protein has stronger anaphylaxis, is not suitable for human body, is only limited to do the makeup use.

The objective of the invention is to provide a kind of method that can produce pure human lysozyme in enormous quantities.

Solution of the present invention is utilization Protocols in Molecular Biology and genetic engineering technique, human lysozyme gene is cloned in the yeast thalline expresses, and makes human lysozyme by extraction, purifying then.

Specifically finish by following steps:

One, obtain human lysozyme gene:

From human leukocyte, extract RNA, after reverse transcription is cDNA, carries out poly chain reaction (PCR) and amplify human lysozyme gene.

Two, expression system:

With carrier for expression of eukaryon pPIC9K and pichia spp host bacterium SMD1168 (his ₄, pep ₄) carry out human lysozyme and express.

Three, extract human lysozyme.

The present invention is owing to utilize the genetically engineered recombinant technology, and the fermentative production human lysozyme can be produced human lysozyme in enormous quantities, has avoided again extracting from tissue being subject to the pathogeny pollution problems, and its product has stable, purified characteristics.

The following examples, accompanying drawing can make those skilled in the art more fully understand the present invention.

Embodiment:

One, obtain human lysozyme gene:

From the peripheral white corpuscle of people, extract cell total rna,, use downstream primer P according to lysozyme gene sequence synthesized primer thing ₂, different in nature primer (P ₁P ₂) carrying out polymerase chain reaction PCR, the PCR product is connected under the effect of T4DNA ligase enzyme and transformed into escherichia coli (Top10) recipient cell with the pGEM-T carrier, is layered on the LB flat board of the blue or green enzyme element of ammonification benzyl 37 ℃ of grow overnight.4 clones of picking are inoculated in respectively in the LB liquid nutrient medium that contains the blue or green enzyme element of ammonia benzyl, 37 ℃ of shaken overnight.Collect thalline,, cut evaluation through enzyme and determine positive colony according to ordinary method a small amount of extracting plasmid.Carry out the nucleotide sequence analysis with dideoxy chain termination, the result is in full accord with people's bacteriolyze sequence of delivering.With Xho I and EcoR I double digestion pGEM-T-hIy plasmid, glue reclaims the hIy gene fragment.With Xho I and EcoR I double digestion pPIC9K expression vector, glue reclaims pPIC9K linearization plasmid DNA.HIy gene fragment and pPIC9K linearization plasmid DNA connect under the effect of T4DNA ligase enzyme and transformed into escherichia coli (Top10) recipient cell, are layered on the LB flat board of the blue or green enzyme element of ammonification benzyl 37 ℃ of grow overnight.4 clones of picking are inoculated in respectively in the LB liquid nutrient medium that contains the blue or green enzyme element of ammonia benzyl, 37 ℃ of shaken overnight.Collect thalline,, cut evaluation through enzyme and determine positive colony according to ordinary method a small amount of extracting plasmid.

Two, recombinant plasmid pPIC9K-hIy makes up:

Recombinant plasmid pPIC9K-hIy makes up and is explained by accompanying drawing.

Three, jigging of reorganization SMD1168-hIy engineering strain:

To recombinate the pPIC9K-hIy plasmid behind the Bg/11 linearization for enzyme restriction, with electroporation it is changed in the pichia spp cell, go out by the transformant of 0.5-5mg/ml G418 (Glucosaminitol antibiotics) pressure screening 5mg/ml G418 is screened, detect the human lysozyme gene stable integration to Pichia yeast karyomit(e) through PCR, this bacterial strain called after Recombinant SMD1168-hIy strain is called for short the human lysozyme gene engineering bacteria.

Four, genetically engineered human lysozyme production technique:

Engineering bacteria is inoculated in the BMGY training base, 30 ℃ of condition bottom fermentations 36 hours, centrifugal receipts bacterium adds in the BMMY training base, and abduction delivering was 30 ℃ of condition bottom fermentations 24-72 hour, centrifugal, collect supernatant liquor, concentrated solution is collected in molecular weight cut-off 10KD ultra-fine filter, ultrafiltration, and through the SP ion exchange column, wash-out is left and taken active peak liquid continuously, identify the packing freeze-drying, be finished product.

Prove that through SDS-PAGE result electrophoresis is a band, its molecular weight of albumen is approximately 15KD, western blot confirms to have the attribute of natural human N,O-Diacetylmuramidase, and the mensuration of antalzyme activity is to lower method with turbidity, than the high 1-3 of commercial chicken antalzyme activity doubly, with dull and stereotyped solusphere method experiment, clinical samples is isolating to the drug-fast bacterium of antibiotic commonly used, all to the human lysozyme sensitivity, having showed to have good application prospects, is a kind of more satisfactory anti-inflammatory, anti-microbial type medicine.

Claims (4)

1, a kind of method of producer gene engineered recombinant human lysozyme, finish by following steps:

One, obtain human lysozyme gene:

From human leukocyte, extract RNA, after reverse transcription is cDNA, carries out poly chain reaction (PCR) and amplify human lysozyme gene;

Two, expression system:

With carrier for expression of eukaryon pPIC9K and pichia spp host bacterium SMD1168 (his ₄, pep ₄) carry out human lysozyme and express;

Three, extract human lysozyme.

2, the method for human lysozyme according to claim 1, the step of obtaining human lysozyme gene is:

From the peripheral white corpuscle of people, extract cell total rna,, use downstream primer P according to lysozyme gene sequence synthesized primer thing ₂, different in nature primer (P ₁P ₂) carrying out polymerase chain reaction PCR, the PCR product is connected under the effect of T4DNA ligase enzyme and transformed into escherichia coli (Top10) recipient cell with the pGEM-T carrier, is layered on the LB flat board of the blue or green enzyme element of ammonification benzyl 37 ℃ of grow overnight; 4 clones of picking are inoculated in respectively in the LB liquid nutrient medium that contains the blue or green enzyme element of ammonia benzyl, 37 ℃ of shaken overnight; Collect thalline,, cut evaluation through enzyme and determine positive colony according to ordinary method a small amount of extracting plasmid; Carry out the nucleotide sequence analysis with dideoxy chain termination, the result is in full accord with people's bacteriolyze sequence of delivering; With Xho I and EcoR I double digestion pGEM-T-hIy plasmid, glue reclaims the hIy gene fragment; With Xho I and EcoR I double digestion pPIC9K expression vector, glue reclaims pPIC9K linearization plasmid DNA; HIy gene fragment and pPIC9K linearization plasmid DNA connect under the effect of T4DNA ligase enzyme and transformed into escherichia coli (Top10) recipient cell, are layered on the LB flat board of the blue or green enzyme element of ammonification benzyl 37 ℃ of grow overnight; 4 clones of picking are inoculated in respectively in the LB liquid nutrient medium that contains the blue or green enzyme element of ammonia benzyl, 37 ℃ of shaken overnight.Collect thalline,, cut evaluation through enzyme and determine positive colony according to ordinary method a small amount of extracting plasmid.

3, the method for human lysozyme according to claim 1, the step that jigs of reorganization SMD1168-hIy engineering strain is:

To recombinate the pPIC9K-hIy plasmid behind the Bg/11 linearization for enzyme restriction, with electroporation it is changed in the pichia spp cell, go out by the transformant of 0.5-5mg/ml G418 (Glucosaminitol antibiotics) pressure screening 5mg/ml G418 is screened, detect the human lysozyme gene stable integration to Pichia yeast karyomit(e) through PCR, this bacterial strain called after Recombinant SMD1168-hIy strain is called for short the human lysozyme gene engineering bacteria.

4, the method for human lysozyme according to claim 1, extraction process is:

Engineering bacteria is inoculated in the BMGY training base, 30 ℃ of condition bottom fermentations 36 hours, centrifugal receipts bacterium adds in the BMMY training base, and abduction delivering was 30 ℃ of condition bottom fermentations 24-72 hour, centrifugal, collect supernatant liquor, concentrated solution is collected in molecular weight cut-off 10KD ultra-fine filter, ultrafiltration, and through the SP ion exchange column, wash-out is left and taken active peak liquid continuously, identify the packing freeze-drying, be finished product.