CN103088000A - Method for overexpression of cholate activation lipase in mammary gland of mammal - Google Patents

Abstract

The invention provides a method for overexpression of cholate activation lipase in mammary gland of a mammal. The method comprises the following steps of transferring the gene coding human cholate activation lipase into the mammal so as to realize overexpression of human cholate activation lipase in the mammary gland of the mammal. According to the method, a transgenic mouse is prepared by utilizing construction of a BSSL (bile salt stimulation lipase) mammary gland specific expression vector; and human cholate activation lipase is overexpressed in the mammary gland of the mouse, the expression quantity of the human cholate activation lipase in milk of the transgenic mouse can achieve 0.38mg/ml, and the activity is 91473.94mU/ml. A premature young mouse is fed with transgenic milk of high expression cholate activation lipase, and the result shows that the survival rate of the premature young mouse is improved by 10% compared with that of the premature young mouse fed with milk of wild mouse. The method lays a foundation for producing human cholate activation lipase by utilizing a large livestock mammary gland bioreactor.

Description

Cross the method that cholate activates lipase of expressing in mammal galactophore

Technical field

The present invention relates to the genetically engineered field, particularly relate to a kind of method that cholate activates lipase of expressing of crossing in mammal galactophore.

Background technology

Cholate activates lipase (Bile salt-stimulated lipase, BSSL) be found to be present in the milk of people, gorilla, cat, dog, ferret, mouse, and do not exist in milk, it is stable that its activity can keep under baby's stomach acidic conditions and pepsin hydrolysis condition, plays very important effect for triglyceride level in newborn infant's intestinal absorption milk.The BSSL activity is stable under the pH of baby's gastric juice environment, and cholate protects it to avoid the Trypsin inactivation, until could be hydrolyzed triglyceride in milk with cholate mixing BSSL in 12 fat intestines.BSSL keeps active reason under stomach acidic conditions and pepsin hydrolysis condition be mainly the glycosylation repetition position of BSSL aerobic connection.Because Saliva Orthana plays a protective role, wherein confirmable is protection small intestine and gastric epithelial opposing acidic conditions and not by protease hydrolysis.With respect to steapsin, BSSL hydrolysis emulsification peptization and water-soluble substrate, and there is no location specific.

BSSL is a kind of secretor type enzyme, mainly expresses in mammary gland and exocrine pancreas in lactation period, is activated by cholate in small intestine.BSSL accounts for 5% of enzyme content in pancreatic juice.The mouse that knocks out BSSL shows that lacking BSSL does not affect small intestine to the absorption of non-esterified fat.But BSSL is unique to the hydrolytic activity of cholesterol ester in digestive tube, and assists for effective hydrolysis of triglyceride level and phosphatide.BSSL is responsible for regulating small intestine to the absorption of cholesterol ester, but does not play a major role for the absorption of free cholesterol.

The BSSL gene of people, rat, mouse conservatively has 11 exons and 10 introns.Each exon has a key structure and/or functional area.For example, 1 exon coded signal peptide, it is crosslinked that 3 exons comprise first intramolecular halfcystine, and second intramolecular halfcystine is crosslinked by 7 exon codings.Avtive spot Serine-194, Histidine-435, aspartic acid-320 are respectively by 5,8,10 exon codings.The ring on surface is covered the groove of avtive spot by 4 exon encoding parts.The exon that conservative property is minimum in different plant species is No. 11, the repeating unit of its coding oxygen linked glycosylation zone and C-terminal proline rich.The most important number of repeat unit purpose difference that is not both proline rich of 11 exon nucleotide sequences.According to former report, the BSSL gene of rat and mouse has respectively 4 and 3 repeating units.Comparatively speaking, people's BSSL gene has the polymorphism of height, the albumen of 16 repeating units of allelotrope coding that great majority are total.High-frequency polymorphism is the existence due to gene 3 ' end hypervariable region.5 ' end sequence has classical TATA and CCAAT element, and is relevant to organizing specific expression.The upstream sequence of BSSL gene also contains AP1, AP2 and SP1 binds the consensus sequence in site.Yet, can't determine that these cis elements are to the effect of regulation and control BSSL genetic expression function aspects.

Summary of the invention

The objective of the invention is by genetic engineering technique, cross the expression cholate and activate lipase in mammal galactophore.

In order to realize the object of the invention, a kind of method that cholate activates lipase of expressing of crossing in mammal galactophore of the present invention, it is that the gene that encoding human source cholate activates lipase is changed in Mammals, activates lipase thereby realize crossing expression people source cholate in mammal galactophore.

Preferably, preceding method is that the gene that encoding human source cholate activates lipase is changed in Mammals by methods such as microinjection, somatic cell clone method or sperm vector methods, activates lipase to realize crossing expression people source cholate in mammal galactophore.

Wherein, people source cholate activates the aminoacid sequence of lipase as shown in SEQ ID No.1, or this sequence is through replacing, lack or adding one or several amino acids formed aminoacid sequence with same function.

Preferably, the above-mentioned people of coding source cholate activates the gene order of lipase as shown in SEQ ID No.2.

Aforesaid method, described Mammals are gorilla, cat, dog, ferret, mouse etc.

The present invention also provides the mammary gland-specific expression vector of the gene that contains encoding human source cholate activation lipase.The carrier that preferably sets out is pBC1.

The present invention also provides the transgene mammal that contains above-mentioned expression vector cell.

The present invention also provides a kind of method for preparing clone embryos, and it is take above-mentioned transgenic cell as the nuclear transplantation donorcells, and stripped ovocyte is the nuclear transplantation recipient cell, obtains clone embryos by nuclear transfer technology.

The present invention further provides a kind of method for preparing breeding transgenic livestock, it is the clone embryos that aforesaid method prepares to be moved into the domestic animal intrauterine by Nonoperative method carry out gestation, obtains breeding transgenic livestock.

The present invention utilizes and builds the BSSL mammary gland-specific expression vector, and the preparation transgenic mice is crossed the cholate of expressing the people and activated lipase in mammary gland of mouse, and its expression amount in transgenic mice milk can reach 0.38mg/ml, and activity is 91473.94mU/ml.The transgenosis milk that utilizes the high expression level cholate the to activate lipase premature labor children mouse of feeding, result show that the survival rate of premature labor mouse the wild mouse milk of feeding has improved approximately 10%.For utilizing from now on heavy livestock galactophore biological reactor production people source cholate activation lipase to lay the first stone.

Description of drawings

Fig. 1 is that pBC-hBSSL carrier linearizing enzyme cuts back to close structure iron.

After Fig. 2 is double digestion pBC-hBSSL carrier, reclaim the electrophoresis result of the purpose fragment that is used for microinjection; Wherein, M is λ/Hind III molecular weight standard; PBC-hBSSL is the linearizing carrier segments of 18kb.

Fig. 3 is the transgenic mice preparation flow.

Fig. 4 is that PCR detects F0 for positive transgenic mice result; Wherein, M is 1kb gradient DNA molecular amount standard; The transgenic mice genome is respectively 4,6,9,13,14,24,25 and the negative contrast of 29, WT, the PCR result of the positive plasmid of P.

Fig. 5 is the integration of foreign gene in Southern hybridization check transgenic mice genome; Wherein, M is 1kb gradient DNA molecular amount standard; WT is the wild-type mice contrast; P1, P5, P10 are respectively the positive plasmid contrast results of hybridization that is equivalent to 1,5,10 copy numbers; 4,6,9,13,14,24,25 and 29 be respectively F0 for the corresponding genomic results of hybridization of positive mouse.

Fig. 6 is feed respectively premature labor and the normal young baby's who produces survival rate situation of the female mouse of transgenosis and wild-type; Wherein, H is people's milk sample results of hybridization; WT is wild-type mice breast sample hybridization contrast; 9,13,14,24,25 and 29 be respectively F0 for positive mouse breast sample results of hybridization.

Fig. 7 is feed respectively premature labor and the normal young baby's who produces survival rate situation of the female mouse of transgenosis and wild-type; Wherein, WN, the normal young baby who produces of the female mouse foster nursing of wild-type; BN, the normal young baby who produces of the female mouse foster nursing of transgenosis; BP, the female mouse foster nursing of transgenosis premature labor young baby; WP, the female mouse foster nursing of wild-type premature labor young baby; Every group is respectively 3 female mouse generation hello 30 young babies altogether.

Fig. 8 is feed respectively premature labor and the normal young baby's who produces body weight gain situation of the female mouse of transgenosis and wild-type; Wherein, WN, the normal young baby who produces of the female mouse foster nursing of wild-type; BN, the normal young baby who produces of the female mouse foster nursing of transgenosis; BP, the female mouse foster nursing of transgenosis premature labor young baby; WP, the female mouse foster nursing of wild-type premature labor young baby.Every group is controlled at 8-10 young baby.From first day until the 21st day of wean, every morning, 9:00-12:00 measured to young baby's body weight determination.

Fig. 9 is feed the respectively situation of premature labor and the normal young baby's who produces ight soil main component of the female mouse of transgenosis and wild-type; Wherein, WN, the normal young baby who produces of the female mouse foster nursing of wild-type; BN, the normal young baby who produces of the female mouse foster nursing of transgenosis; BP, the female mouse foster nursing of transgenosis premature labor young baby; WP, the female mouse foster nursing of wild-type premature labor young baby.

Embodiment

Following examples are used for explanation the present invention, but are not used for limiting the scope of the invention.If do not specialize, the conventional means that in embodiment, technique means used is well known to those skilled in the art.

The carrier that following examples are used, reagent and source thereof:

PBC1 carrier (Invitrogen) contains the IRATp970H1068D carrier of human lipoprotein lipase cDNA sequence available from Imagenes company.The synthetic sequencing that reaches of primer is completed by the living work in Shanghai.Laboratory animal is used the Kunming small white mouse, available from Beijing Experimental Animal Center.Taq enzyme, T4DNA ligase enzyme, restriction endonuclease are Biolabs company product; Plasmid purification and to reclaim test kit be Omega company product; The isotope labeling reagent box is available from Promega company; Radio isotope α- ³²P-dCTP and Southern Hybond membrane are available from U.S. Amershambiosciences company.The primary antibodie of anti-hBSSL is available from Santa Cruz company.BSSL content detection ELISA test kit is available from GBD Corp..Triglyceride level, cholesterol, glucose detection test kit be available from the northern control of middle life company, and the BCA protein detection kit is available from green skies company.The conventional molecular biology experiment operation stepss such as enzyme is cut, connected, recovery, conversion, pcr amplification see " molecular cloning (third edition) " for details.

Embodiment 1 people source BSSL mammary gland expression vector builds

Obtain people source BSSL gene cDNA sequence take the IRATp970H1068D plasmid as the template pcr amplification.The pair of primers that is used for amplification cDNA sequence is hBSSL-F (5 '-CCGCTCGAGACCATGCTCACCATGGGGCGC-3 ') and hBSSL-R (5 '-CCGCTCGAGTTCAGGGGTATGAGGCTTTAT-3 '), introducing XhoI restriction enzyme site in primer respectively.Extension increasing sequence is reclaimed, be cloned in the pBC1 carrier of cutting through the XhoI enzyme (Fig. 1), identify Insert Fragment sequence and direction by PCR, and check order, insert the most at last correct expression vector called after pBC-hBSSL.

Foundation and the detection of embodiment 2 transgene mouse models

1.1 transgenic mice is produced in microinjection

Studies show that, produce transgenic mice with microinjection, linear DNA is higher than cyclic DNA integration efficiency, therefore the expression vector pBC-hLPL enzyme that builds will be cut except carrying out microinjection after useless protokaryon part linearizing simultaneously.

Cut the expression vector pBC-hBSSL that builds with the NotI/SalI enzyme, remove prokaryotic vector skeleton structure part, then enzyme is cut DNA and carried out agarose gel electrophoresis, downcut the gel at the place, 18kb position that contains the purpose fragment to be recycled, utilize the gel recovery test kit (Omega) of the above DNA fragmentation of recyclable 10kb to reclaim, result as shown in Figure 2.

To reclaim fragment and be dissolved in TE solution (10mmol/L Tris.cl (pH7.4), 0.1mmol/LEDTA, with the preparation of Milli-Q water) after, measure OD ratio and calculating concentration under 260nm and 280nm in ultraviolet spectrophotometer, the OD260/OD280 ratio of above-mentioned two kinds of purification process can be used for injecting mouse fertilized egg all more than 1.8.To contain the TE solution dilution of exogenous genetic fragment to final concentration 2.5ug/ml, be expelled to the male pronucleus of mouse fertilized egg, then be transplanted in the female mouse uterine tube of false pregnancy of estrus synchronization.The transgenic mice preparation flow as shown in Figure 3.

1.2 the PCR of transgenic mice detects

Get 3 age in week transgenic mice tail organize sample, extract DNA.Take the genome that extracts as template, utilize people source BSSL cDNA sequences Design primer (L-F:5 '-GATTGACAAGTAATACGCTGTTTCCTC-3 ', L-R:5 '-ACAGGTAGTTGTTGAGGAAGTT-3 ') can amplify the fragment of 499bp size in the transgenic positive mouse, with the negative contrast of wild-type mice genome, with the positive contrast of carrier pBC-hBSSL.Amplification condition is 94 ℃, 30sec; 58 ℃, 30sec; 72 ℃, 90sec; 30 circulations.1.0% agarose electrophoresis observations.PCR detects 8 positive mouse in F0 generation 42 mouse, result is the transgenic positive mouse the 4th, 6,9,13,14,24,25 and No. 29 as shown in Figure 4.

1.3 the Southern blotting of transgenic mice detects

F0 generation 8 transgenic mices with the PCR test positive, cut afterbody tissue extraction genome, getting 10 μ g digests with the PstI restriction endonuclease, the negative contrast of wild-type mice genome of cutting with the PstI enzyme, the positive contrast of pBC-hBSSL injection carrier segments that the PstI enzyme is cut, hybridization probe is to utilize the gene fragment (primer is S-F:5 '-AGACCCTCTCCCCCTACAACAA-3 ', S-R:5 '-GGCATATGAGCTTCCTTGGAGT-3 ') that reclaims 1.1kb after pcr amplification take people BSSLcDNA as template.With sample low pressure electrophoresis at a slow speed, adopt the alkali transfer method that DNA is transferred to nylon membrane from agarose, the one side that nylon membrane is had a DNA is inwardly, be rolled into tubular and put into hybrid pipe, add prehybridization solution, be not shorter than 1hr in 65 ° of C prehybridizations, add the α-P through heat denatured ³²The isotope-labeled probe of dCTP, 65 ° of C hybridization, 12 ~ 16hr wrap with preservative film after washing film, put in magazine, press X-ray in the darkroom, in-70 ° of C radioautograph, rinse the X-ray observations in 12 hours.Southern blotting result as shown in Figure 5, result shows that positive mouse Southern blotting detected result is consistent with the PCR detected result.

The expression analysis of embodiment 3 recombinant human B SSL in transgenic mice breast sample

1.1 the Western blotting of transgenic mice breast sample detects

Gather F0 generation 9,13,14,24, the 25 and No. 29 mouse lactation milk sample in mid-term, wild-type mice milk is as negative control, the positive contrast of people's milk.Milk gathered after mouse birth in 8-12 days, before gathering, female mouse separated more than 3 hours with newborn mouse, and to the pitocin of abdominal injection doses.Centrifugal rear removal butterfat and caseic whey are being forwarded on nylon membrane after electrophoresis on 10% SDS-PAGE.Carry out WesternBlot with anti-human BSSL primary antibodie and analyze, can detect the expression of recombinant human B SSL in transgenic mice milk.Recombinant human B SSL is consistent with BSSL molecular size range in people's milk, is about 100kDa, and result as shown in Figure 6.

1.2 BSSL expression analysis in transgenic mice breast sample

Adopt the ELISA method BSSL expression amount in transgenic mice milk is analyzed, the milk sample of having chosen 6 transgenic mice lactation mid-terms detects.Owing to adopting business-like ELISA test kit, can not effectively distinguish the BSSL in people source and mouse source, thereby the present invention's milk sample (concrete operations by specification) in contrast of having detected 6 wild-type mice lactation mid-terms.The BSSL content that transgenic mouse is expressed is significantly higher than the BSSL content in wild-type mice milk, and high expression level amount reaches 0.38mg/ml(table 1).

1.3 in the transgenic mice milk sample, the activity of BSSL detects

Adopt and carry out same a collection of transgenosis that the BSSL expression analysis uses and the mouse milk of wild-type and carry out the detection of enzymic activity.Use contains ³The glycerine emulsion of H mark triolein is substrate, by extracting, glyceryl ester and free fatty acids are separated after 37 ℃ of incubation reaction of sample and substrate, it is active that the labelled with radioisotope method that the radioactivity of the free fatty acids that steatolysis produces is directly proportional to the activity of lipase in sample is measured external BSSL.Concrete operations can be with reference to Zhang Aihong, Liu Guoqing, the labelled with radioisotope method detects lipoprotein lipase activity and application thereof).Detected result is as shown in table 1.

Table 1 transgenic mouse milk and wild-type mice milk are at lactation BSSL in mid-term expression amount and active cartogram

*P<0.05;***P<0.001。Above data represent mean value

The detection of embodiment 4 transgenic mouse milk to premature labor children mouse growth effect

1.1 the detection to premature labor young baby survival rate

For feeding premature labor and the normal young baby who produces, all young babies are not all that breeder mother feeds to the female mouse of transgenosis and wild-type respectively, and every female mouse does not carry out sex and distinguishes for feeding to such an extent that the young baby only is controlled at 8-10 between the young baby.The normal mouse that produces derives from spontaneous labor, and the premature labor mouse obtains by the pregnant mouse c-section to conceived 19 days.

The normal young baby who produces feeds in the female mouse of transgenosis or wild-type generation, and its survival rate is 100%.Be 93.3% higher than 83.3% (Fig. 7) that is fed by the female mouse of wild-type generation and the survival rate of premature labor mouse was fed by the female mouse of transgenosis generation.

1.2 the detection to BSSL activity in premature labor young baby stomach

It is to guarantee that BSSL is not degraded by digestive ferment in stomach and guarantees that it is active in young mouse digestive tube that the activity under one's belt of BSSL in breast milk is detected.After birth the 10th day, feed BSSL in the stomach of transgenosis milk of the normal mouse that produces was significantly higher than the premature labor mouse transgenosis milk of feeding.The premature labor mouse is by being fed by the female mouse of wild-type generation of feeding of the female mouse of transgenosis generation, and in stomach content, the BSSL activity increased by 108% at the 10th day, had increased 73%(table 2 at the 20th day).

Rear the 10th day and the 20th day of table 2 birth, in normal production and premature labor young baby stomach, the BSSL of breast milk is active

*P<0.05;**P<0.01。N is the laboratory animal number.

The mensuration of front body weight gain 1.3 the premature labor young baby weans

Premature labor and the normal connatae body weight average of young baby of producing are respectively at 1.50g and 2.06g.As seen from Figure 8, the body weight gain that the premature infant that transgenosis milk is fed feeds than wild-type is remarkable, and the 21st day of wean, BP group and WP group young baby's body weight average was respectively 12.38 and 8.61g, and BP organizes and increased by 43.79% than WP.

The mensuration of main component in front ight soil 1.4 the premature labor young baby weans

Mensuration to main component in ight soil is in order to analyze the loss situation of mouse nutritive ingredient.Main component in young baby's ight soil of lactation (fat, protein, dry-matter) is measured, each composition is with (* * P＜0.01 as shown in Figure 9 of percentage in ight soil, * P＜0.05), the content of fecal fat, the BN group is 71.16% (P＜0.01) in organizing for WN; The BP group is 67.66% (P＜0.05) of WP group.There is no significant difference between each group of the content of albumen and dry-matter in ight soil.

Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements, all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.

Claims (10)

1. cross in mammal galactophore and express the method that cholate activates lipase, it is characterized in that, it is that the gene that encoding human source cholate activates lipase is changed in Mammals, activates lipase thereby realize crossing expression people source cholate in mammal galactophore.

2. method according to claim 1, is characterized in that, it is that the gene that encoding human source cholate activates lipase is changed in Mammals by microinjection, somatic cell clone method or sperm vector method.

3. method according to claim 1 and 2, it is characterized in that, people source cholate activates the aminoacid sequence of lipase as shown in SEQ ID No.1, or this sequence is through replacing, lack or adding one or several amino acids formed aminoacid sequence with same function.

4. method according to claim 3, is characterized in that, the gene order of encoding human source cholate activation lipase is as shown in SEQ ID No.2.

5. method according to claim 1 and 2, is characterized in that, described Mammals is gorilla, cat, dog, ferret, mouse.

6. the mammary gland-specific expression vector that contains the gene of encoding human source cholate activation lipase.

7. expression vector according to claim 6, is characterized in that, its carrier that sets out is pBC1.

8. the transgene mammal cell that contains claim 6 or 7 described expression vectors.

9. method for preparing clone embryos, it is take transgenic cell claimed in claim 8 as the nuclear transplantation donorcells, and stripped ovocyte is the nuclear transplantation recipient cell, obtains clone embryos by nuclear transfer technology.

10. method for preparing breeding transgenic livestock, it is that clone embryos with the described method preparation of claim 9 moves into the domestic animal intrauterine by Nonoperative method and carries out gestation, the acquisition breeding transgenic livestock.

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