CN101240292A - Construction of human paraoxonase 3 gene expression vector - Google Patents
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Abstract
The invention belongs to biotechnology pharmacy technology field. The recombination plasmid Pci-Hpon3 expressed in zooblast is constructed by used human mintacol enzyme 3 gene (Hpon3), Hpon3 is expressed efficiently and secreted into blood in small murine skeletal muscles organizing in electric transferring method to improve PON3 in blood serum and enforce the ability of small mouse antioxidation pressure so that it can control liver damage efficiently. The activity of Hpon3 transgene expression small mouse blood serum hydrolytic decomposition melilotol is improved by 1.4 times and can maintain 24 days. After small mouse acute or subacute liver damage mould is built by injecting carbon tetrachloride into abdominal cavities of transgene group and normal small mouse, the blood serum ALT and AST level of transgene small mouse are lower greatly, malondialdehyde (MDA) level of liver homogenate is lower, reducing glutathione (GSH) and total antioxidant ability (T-AOC) are higher than contrasting group, and transgene group small mouse liver cell damage extent indicated in nosology slicer result is lower than CC1(4) control group's indicate that recombination human PON3 plasmid transgene expression can improve efficiently blood serum PON3 activity and reduce liver oxygenizing pressure so that it can be used for preventing and treating liver damage.
Description
One. technical field
The invention belongs to the biotechnological pharmaceutics technical field.
Two. background technology
Paraoxonase 3 (PON3) is that the paraoxonase gene family is found the latest, studies minimum member.People PON3 gene cDNA total length 1075bp, the head of district 1065bp that wherein encodes, 354 amino acid of encoding.Gene expression product PON3 is the glycoprotein of the about 40kDa of molecular weight, is a kind of calcium ion dependency esterase, has aromatic ester enzymic activity, lactonase activity and anti-oxidant function [Lu H Q, et al.Biochem Pharmaco, 2005,70:1019-1025].People PON3 mainly goes into blood by the liver cell synthesis secretion, and 98% combines with HDL in serum, but the content of PON3 only is 1/50 of PON1 in the serum.Experimental results show that; PON3 not only has the effect that suppresses HDL and LDL oxidative modification; and stronger resistance of oxidation arranged; the rabbit anteserum PON3 of purifying is at strong 100 times of [the Draganov DI of energy force rate PON1 of the anti-cupric ion inductive of external protection LDL oxidative modification; et al.JBiol Chem, 2000,275:33435-33442]; therefore present most research concentrates on PON3 and reduces intravital oxidative pressure, prevents and treats the atherosclerosis aspect.
Existing studies show that, the rising of oxidative pressure and lipid super-oxide is the common pathomechanism of multiple hepatic diseases, and it is the important paathogenic factor of liver injury that the oxidative pressure rising causes the living radical increase, and therefore anti-oxidant treatment can be applicable to the liver injury control.We once found: muscle electrotransfer mediation external source hPON1Q expression of gene and secretion; effectively reduce the oxidative pressure of liver; chmice acute liver injury to tetrachloro-methane induction has good provide protection [Qin Jun river etc., number of patent application: 200610097968.6].Because PON3 shows the antioxygenation stronger than PON1; and document does not see that PON3 is applied to the relevant report of hepatic diseases prevention; we have studied with transgene expression and have improved the activity of blood-serum P ON3 to reduce oxidative pressure; the protection liver, this may be a kind of approach of new control liver injury.
Tetracol phenixin single injection inducing mouse acute liver damage is a kind of widely used screening and the model of estimating the liver protecting medicine, the subacute liver injury model of injection tetrachloro-methane induction is used to assess the effect of electrotransfer hPON3 anti-liver injury next day that the present invention having set up repeatedly simultaneously, this model is compared the generating process that more approaches clinical hepatic diseases with the acute liver damage model, therefore has more practical significance.
China is hepatic diseases country occurred frequently, and liver injury that viral hepatitis, alcohol and drug intoxication hepatopathy etc. cause and consequent hepatic fibrosis, liver failure etc. have become one of common disease.Traditional hepatic comprises antioxidant, the endogenous protection factor, anti-cholestasis medicine and part plant amedica etc.But they have mostly action time short, onset is slow, need for a long time shortcoming such as repeatedly medication, utilizing gene therapy methods to import and protecting the liver gene medicine and make it to efficiently express for a long time with prevention and treatment hepatic diseases has become a kind of strategy that the applications well prospect is arranged.
As one of method of transgenosis, electricity irritation can mediate exposed plasmid DNA efficiently expressing in various kinds of cell and tissue.Compare with virus vector, this technology has plurality of advantages such as biological safety is good, experimental procedure simple, saving cost.The skeletal muscle tissue cell quantity is huge; be easy to carry out various operations; forming cell mostly is sophisticated multinuclear myocyte and upgrades very slow; blood supply is abundant, and the albumen synthesis capability is strong, and the normal secretion of the albumen mass-energy of cell expressing enters blood circulation; these characteristics determined its can be used as target tissue [the McMahon JM of ideal transgenosis; et al.Biodrugs, 2004,18:155-165].The report proof is arranged, and the electricity irritation mediation expression level of foreign gene in skeletal muscle tissue down improves 2-4 the order of magnitude than traditional non-virus carrier transfer method, and the longest sustainable expression of gene is more than 9 months.We use the YC-2 type programmable stimulato that Chengdu Instruement Factory produces, and connect stainless steel needle-like electrode, have groped suitable electrical stimulation parameters, have reached electrotransfer effect preferably.
Three. summary of the invention
The problem that the present invention need solve is the mammalian cell expression vector that makes up recombinant human paraoxonase 3 genes; the method of utilizing electricity irritation mediation change it over to mice skeletal organizationally efficient expression and by self N end signal peptide continuous release improving the activity of blood-serum P ON3, thereby as the antagonism of novel gene medicine by the hepatocellular injury of tetrachloro-methane induction to protect liver effectively.
Technical scheme of the present invention comprises: 1. design and the structure of recombinant human paraoxonase gene 3 mammalian cell expression vector pCI-hPON3.2. electricity irritation mediation pCI-hPON3 efficiently expressing and secrete in mice skeletal.3. recombinant human PON3 can improve serum hPON3 level as genomic medicine, is applied to prevent and treat liver injury.
1.pCI-hPON3 design and structure
The structure flow process of pCI-hPON3 plasmid as shown in Figure 1.With pMD18T-hPON3 is template, with primer 1,2 total length hPON3 gene orders through PCR method amplification 1065bp, and it is cloned between the EcoR I and SalI site of pCI carrier (available from Promega company), the hPON3 gene is under the control of CMV promotor, obtains recombinant plasmid pCI-hPON3.Through the order-checking proof, the gene order that obtains is consistent with design.PCI-hPON3 plasmid CaCl
2Method changes in the intestinal bacteria and increases, and a large amount of productive rates that extract of plasmid are 3.15 μ g/ml intestinal bacteria, OD260/280=1.82.
Primer 1:5 ' AAGAATTCATGGGGAAGCTCGTGGCG 3 '
Primer 2: 5 ' CCGTCGACCTAGAGCTCACAGTACAG 3 '
2. the PON3 skeletal muscle of electricity irritation mediation is expressed
The pCI-hPON3 plasmid of 25 μ l, 2 μ g/ μ l is injected in the male ICR mouse left side shin bone flesh of 10 anesthesia in advance.After 30 seconds, the needle electrode of two access electrical stimulators is inserted the both sides, injection site along the myofiber direction carry out the operation of gene electrotransfer, electric pulse field parameter see claim 2.The pCI plasmid of unloaded plasmid control group mice intramuscular injection equal in quality also gives the electricity irritation of the same terms, and the PBS control group mice is only injected 25 μ l PBS.Each organizes the activity of getting blood and centrifugation 5 μ l determination of serum hPON3 hydrolysis melilotine in per two days from corner of the eyes vein after the injected in mice.Electricity changeed after 24 hours, and the muscle tissue 10mg that unloaded plasmid control group and transgenosis group mouse are got liver and electrotransfer position respectively extracts total RNA, was RT-PCR with primer 3,4, with the expression of pCI-PON3 in muscle of determining that electricity changes.RT-PCR the results are shown in accompanying drawing 2.The expression of mouse β-actin is as confidential reference items.
Primer 3:5 ' ATACTGTGTATCTTTATGTTGTG 3 '
Primer 4:5 ' TGAAGCACAGAGCCATTGTTG 3 '
Recombinant plasmid pCI-hPON3 shifts to enter under the electricity irritation mediation and expresses hPON3 albumen in the mice skeletal tissue, and the hPON3 protein excretion of biologically active is to blood.Transgenosis group mice serum PON3 activity was compared with two control groups behind the injection plasmid and is begun to raise in 24 hours, reached maximum (2.40 ± 0.05U/ml) on the 8th day, near PBS control group blood-serum P ON3 activity (1.69 ± 0.11U/ml) and pCI control group blood-serum P ON3 activity (1.4 times (P＜0.05) of 1.69 ± 0.12U/ml), sustainable at least 24 days of high-caliber expression, two control group mice blood-serum P ON3 activity do not have considerable change during this period, illustrate that the hPON3 gene can enter blood circulation by the continuous expression justacrine under the electric shock mediation in the mice skeletal tissue.RT-PCR result shows, PON3 level and control group that mouse liver is expressed do not have significant difference, then there is hPON3 specific expressed in the muscle tissue of transgenic mice, unloaded plasmid control group mice muscle does not have expression, the result confirms that further the hPON3 gene has obtained effective expression at the mice skeletal tissue under the electricity irritation mediation, justacrine enters blood circulation, has improved the PON3 activity of mice serum.
3.PON3 recombinant plasmid is applied to prevent and treat liver injury
Transgenosis group injected in mice recombinant plasmid and electricity irritation be after 2 days, abdominal injection 10%CCl
4Semen Maydis oil solution (1ml/kg b.w.) is to set up the chmice acute liver injury model; Other gets 10 source together of the same age male mices abdominal injection same dose CCl every other day
4Semen Maydis oil solution 30 days totally 15 times is to set up subacute liver injury model.Acute and subacute liver injury group is got 10 health source together of the same age male mice injection same dose same number tetracol phenixin respectively as positive control simultaneously, and 10 injected in mice same number 1ml/kgb.w. Semen Maydis oils are as negative control in addition.Last injection CCl
4After 24 hours, pluck eyeball and get blood and put to death mouse, measure mice serum ALT, the AST level, liver homogenate mda (MDA) respectively organized, reduced glutathion (GSH) and total antioxidant activity (T-AOC), and make liver tissue slices to observe the degree of impairment of liver.
Acute and subacute liver injury CCl
4Control group mice Serum ALT, AST compare remarkable rising (P＜0.01) with the normal control group, the MDA level of its liver homogenate is compared raise respectively 1.7 times and 2.3 times (P＜0.05) with the normal control group, and GSH and T-AOC level all have remarkable reduction (P＜0.05).CCl
4The control group mice liver tissue slices is compared with the normal control group, necrocytosis area, cavity, fat drip and the ratio of pathological change such as oedema all significantly increases, the oxidation level that mouse liver is described raises, and acute and subacute liver injury model is set up success respectively.
Behind the acute liver damage group injection tetracol phenixin, transgenosis group mouse and CCl
4Control group is compared Serum ALT and is reduced by 30%, and AST reduces by 32%, significant difference (P＜0.05); The MDA level and the CCl of its liver homogenate
4The control group ratio has reduced by 53%, has recovered near normal control group level (P＜0.05); GSH and T-AOC level then show obvious rising, return to normal level (P＜0.05).Pathological analysis demonstration transgenic mice hepatic necrosis cell area has been compared with positive controls extremely significantly and has been reduced, and the degree of oedema, cavity and steatosis also obviously alleviates.Subacute experiment also shows identical trend: transgenosis group and CCl
4Control group is compared ALT and has been reduced by 80%, and the AST level has reduced by 79%, and MDA, GSH and T-AOC also all have been returned to the level of normal control group.Pathological section shows that inflammatory cell infiltration and liver cell oedema degree around the central Venule obviously alleviate, and the disorderly phenomenon of hepatic necrosis and hepatic cords structure disappears.Respectively organize the variation of mice serum ALT, AST behind the acute liver damage group injection tetracol phenixin and see accompanying drawing 3; Fig. 4 is seen in subacute liver injury group ALT, AST variation; Acute and subacute mouse liver typical case pathological section photo is seen accompanying drawing 5, Fig. 6 respectively.After the above presentation of results transgenosis; high-caliber blood-serum P ON3 can effectively reduce the liver oxidative pressure; acute and subacute hepatocellular injury to tetrachloro-methane induction all has the better protecting effect, so hPON3 can be applicable to preparation prevention and treatment hepar damnification medicine.
The present invention compared with prior art, its beneficial effect is that the present invention is the mammalian cell expression vector pCI-hPON3 that target makes up people's paraoxonase 3 genes with the control liver injury first, through the electricity irritation mediation it is imported in the mice skeletal tissue, and obtain secretion expression people's paraoxonase 3.After the abdominal injection tetracol phenixin is set up chmice acute and subacute liver injury model; detect by the anti-oxidant experiment of liver cell in serum biochemistry index determining and liver organization pathological analysis and the body, confirm that 3 pairs of hepatocellular injuries of people's paraoxonase of transgene expression have significant protective effect.Therefore the present invention is very fast to control liver injury onset, and it is longer that single administration can be kept effective drug duration, and toxic side effect is little, and is with low cost.
Four. description of drawings
The structure schema of Fig. 1 people's paraoxonase gene 3 mammalian cell expression vectors
The RT-PCR analytical results that Fig. 2 transgenosis group and control group mice PON3 express in liver and muscle
(1) control group mice liver
(2) transgenosis group mouse liver
(3) transgenosis group mouse muscle
(4) control group mice muscle
Respectively organize the variation of mice serum ALT, AST after Fig. 3 acute liver damage modeling success
Respectively organize the variation of mice serum ALT, AST after the subacute liver injury modeling success of Fig. 4
Respectively organize mouse typical case hepatopathy photo of science after the modeling of Fig. 5 acute liver damage
(A) normal control group mouse
(B) CCl
4Control group mice
(C) transgenosis group mouse
Respectively organize mouse typical case hepatopathy photo of science after the subacute liver injury modeling of Fig. 6
(A) normal control group mouse
(B) CCl
4Control group mice
(C) transgenosis group mouse
Five. embodiment
1.pMD18T-hPON3 plasmid is made up and is preserved by this chamber.With pMD18T-hPON3 is template, is total to 1065bp with primer 1,2 through PCR method amplification hPON3 full-length gene, inserts in the corresponding multiple clone site of pCI plasmid behind EcoR I and SalI double digestion.The hPON3 gene is under the control of CMV promotor.Through order-checking proof, result and implementation sequence (flow process is seen accompanying drawing 1) in full accord.With pCI-hPON3 conversion CaCl
2The competence TOP10 intestinal bacteria of method preparation, and according to the operating process amplification intestinal bacteria of " molecular cloning " third edition, the PEG method is carried out a large amount of extractions of plasmid.With the purity and the productive rate of plasmid that determined by ultraviolet spectrophotometry extracts, and plasmid concentration is adjusted into 2 μ g/ μ l with PBS.All biochemical reagents are all available from Sigma company, and enzyme is available from Takara company.
2. 30 of healthy male ICR mouses, the SPF level, in age in 4-6 week, body weight 18-22 gram is available from Nanjing Medical University's Experimental Animal Center.Animal is divided into 3 groups at random, is respectively the transgenosis group, empty plasmid control group and PBS control group, 10 every group.Test preceding 1 day according under save described method and measure and respectively organize the active basic value of mice serum PON3 hydrolysis melilotine.Test and respectively organized mouse peritoneal injection Veronal sodium 300mg/kg b.w. in preceding 20 minutes and anaesthetize.With the Hamilton syringe pCI-hPON3 plasmid intramuscular injection of 25 μ l, 2 μ g/ μ l is gone in the left side shin bone flesh of transgenosis group mouse.After 30 seconds, two stainless steel needle-like electrodes insertion both sides, injection site that electrical stimulator connects, interelectrode distance 0.5cm will be inserted.Electricity irritation is taken place by the YC-2 type programmable stimulato that Chengdu Instruement Factory produces, and electric pulse field parameter is: square wave, and 200V/cm, the wide 20ms of ripple, frequency is 1Hz, umber of pulse 8.The pCI plasmid of empty plasmid control group mice injection equal in quality also gives identical electrical field stimulation.The PBS control group is intramuscular injection 25 μ l PBS only.All mouse are all raised in SPF level experimental animal room, free diet, 22 ± 2 ℃ of envrionment temperatures, humidity 45-75%.
The active mensuration of blood-serum P ON3: after testing preceding 1 day and testing per two days, from mouse corner of the eyes venous blood collection, 2000rpm * 10 are minute with separation of serum with the trace blood pipe.Get 5 μ l serum and add 1ml reaction system and abundant mixing.Reaction system comprises 50mM Tris/HCl (pH8.0), 1mM CaCl
2And 1mM melilotine.React in 37 ℃ of water-baths and carried out 10 minutes, add 100 μ l 0.1M EDTA and stop.Assaying reaction liquid is in the photoabsorption at 270nm place and calculate the enzymic activity of PON3 in the serum by the molar extinction coefficient of hydrolysate.Use the variance analysis of SPSS software and relatively the active numerical value of each group mice serum PON3 is carried out statistical analysis in twos.
3. transgenosis group mouse and unloaded plasmid control group mice be after electrotransfer was implemented 24 hours, gets liver organization respectively and electrotransfer muscle position 10mg extracts total RNA, and the pollution of DNA is avoided in DNaseI digestion.Do the RT-PCR reaction with primer 3,4 mentioned above after using the M-MLV reverse transcription, the expression level of mouse β-actin is confidential reference items.The PCR reaction conditions is as follows: 94 ℃ of initial sex change 5min, and 30 circulations, each 94 ℃ of sex change 40s that circulate, 50 ℃ of annealing 45s, 72 ℃ are extended 1min, and 72 ℃ are extended 7min after the loop ends, are stored in-20 ℃.1.2% sepharose isolation identification PCR product.
4. transgenosis group injected in mice recombinant plasmid and electricity irritation pneumoretroperitoneum injection in 2 days gives 10% tetracol phenixin Semen Maydis oil solution 1ml/kg b.w. and induces acute liver damage, and other gets 10 source together of the same age male mices abdominal injection same dose CCl every other day
4Semen Maydis oil solution 30 days is to set up subacute liver injury model.Simultaneously acute and subacute liver injury group is got the identical mouse in two groups of each 10 sources, sex, all ages respectively in contrast.Wherein one group of abdominal injection same dose same number tetracol phenixin is as positive control, and another group abdominal injection same dose same number Semen Maydis oil is as normal control.Last injection CCl
4After 24 hours, each group mouse is plucked eyeball get blood and centrifugation serum, measure Serum ALT, AST level, get liver and make homogenate mensuration MDA, GSH and T-AOC level, clip liver main lobe in addition, place neutral formalin solution to soak and fix 24 hours, after gradient concentration spirituous solution dehydration and dimethylbenzene clean, the paraffin-embedded tissue piece, and be cut into the tissue slice that thickness is 4 μ m with slicing machine.Section is dyeed with h and E, observes, takes pictures under inverted microscope and carry out pathological analysis.Experimental data all adopts variance analysis and relatively carries out statistical analysis in twos.All experimentation on animals operations are all in accordance with " Jiangsu Province's management of laboratory animal regulations ".
Claims (3)
1. the paraoxonase 3 of should choosing is the recombinant plasmid of expressing in mammalian cell that hPON3 makes up, it is characterized in that hPON3 gene clone with pcr amplification between the EcoR of pCI carrier I and SalI site, is positioned under the control of CMV promotor the hPON3 gene.
According to the constructed recombinant plasmid pCI-hPON3 of claim 1 under the electricimpulse mediation in mice skeletal tissue great expression also effectively secrete transgeneic procedure method to blood, it is characterized in that: after mouse shin bone flesh is gone in the pCI-hPON3 plasmid intramuscular injection of 25 μ l, 2 μ g/ μ l, the stainless steel needle-like electrode of two access electrical stimulators is inserted the both sides, injection site, electric pulse field parameter is: square wave, 200V/cm, the wide 20ms of ripple, pulse number 8, frequency is 1Hz.
3. according to the application of the described recombinant expression plasmid of claim 1 in preparation prevention and treatment hepar damnification medicine.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105602989A (en) * | 2012-01-20 | 2016-05-25 | 中国农业科学院上海兽医研究所 | Recombinant vector and application thereof in preparing or screening anti-influenza drugs |
| CN114437179A (en) * | 2022-02-18 | 2022-05-06 | 南京医科大学 | Polypeptide TAC for establishing mammal chronic phospholipid metabolic abnormality model and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105602989A (en) * | 2012-01-20 | 2016-05-25 | 中国农业科学院上海兽医研究所 | Recombinant vector and application thereof in preparing or screening anti-influenza drugs |
| CN105602989B (en) * | 2012-01-20 | 2019-04-02 | 中国农业科学院上海兽医研究所 | A kind of recombinant vector and its application in preparation or screening Tamiflu |
| CN114437179A (en) * | 2022-02-18 | 2022-05-06 | 南京医科大学 | Polypeptide TAC for establishing mammal chronic phospholipid metabolic abnormality model and application thereof |
| CN114437179B (en) * | 2022-02-18 | 2023-05-23 | 南京医科大学 | Polypeptide TAC for establishing mammal chronic phospholipid metabolic abnormality model and application thereof |
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