CN111575316A

CN111575316A - hKLK1 recombinant plasmid, high-expression hKLK1 lentiviral particle, and construction method and application thereof

Info

Publication number: CN111575316A
Application number: CN202010427574.2A
Authority: CN
Inventors: 秦又发
Original assignee: Dongguan Songshanhu Central Hospital Dongguan Shilong People's Hospital Dongguan Third People's Hospital Dongguan Cardiovascular Disease Research Institute
Current assignee: Dongguan Songshanhu Central Hospital Dongguan Shilong People's Hospital Dongguan Third People's Hospital Dongguan Cardiovascular Disease Research Institute
Priority date: 2020-05-19
Filing date: 2020-05-19
Publication date: 2020-08-25

Abstract

The invention provides an hKLK1 recombinant plasmid, a lentiviral particle with high expression level of hKLK1, a construction method and application thereof, wherein the hKLK1 recombinant plasmid is an expression plasmid for cloning hKLK1 gene, and the gene sequence of the hKLK1 recombinant plasmid is shown as SEQ ID: 1. The hKLK1 recombinant plasmid (pEZ-Lv105-hKLK1) can be used for preparing medicaments for treating viral myocarditis and myocardial fibrosis. The lentivirus is used as a vector, and the gene recombination technology is utilized to construct the lentivirus particles of high-expression hKLK1, so that the lentivirus particles are safe and nontoxic, have the function of inhibiting VMC myocardial fibrosis, can meet the use requirement, and have simple construction method, easy operation and good application prospect.

Description

hKLK1 recombinant plasmid, high-expression hKLK1 lentiviral particle, and construction method and application thereof

Technical Field

The invention relates to a therapeutic drug for viral myocarditis, in particular to an hKLK1 recombinant plasmid for inhibiting viral myocarditis myocardial fibrosis, a lentiviral particle with high expression of hKLK1, a construction method and application thereof.

Background

Viral Myocarditis (VMC) is one of the common diseases of the cardiovascular system, and epidemiological investigations have shown that about 15-25% of VMC patients develop myocardial fibrosis and evolve into dilated cardiomyopathy, with a two-year mortality rate of up to 50%. Therefore, the inhibition of the VMC myocardial fibrosis has important significance for maintaining the cardiac structure, improving the cardiac function and reducing the fatality rate. At present, various medicines such as tranilast, carvedilol and captopril can inhibit myocardial fibrosis of VMC mice, but the clinical curative effect of the medicines is not confirmed.

According to published literature reports and clinical treatment experiences, the 3 medicines only improve the pathology of the myocardial fibrosis of the VMC mice, the improvement of the cardiac function is not obvious, and the medicines are rarely used for treating the VMC myocardial fibrosis clinically. The reasons for the above disadvantages are: the target of action of these drugs is not a critical mechanism for VMC myocardial fibrosis.

Disclosure of Invention

Based on the problems, the invention aims to provide an hKLK1 recombinant plasmid, a lentivirus particle with high expression of hKLK1, a construction method and application thereof, wherein the recombinant plasmid and the virus particle can play the roles of inhibiting VMC myocardial fibrosis and improving cardiac function, and have application prospects of being developed into medicines.

In order to achieve the purpose, the invention provides a hKLK1 recombinant plasmid which is a recombinant plasmid for cloning hKLK1 gene, and the gene sequence is shown as SEQ ID: 1.

The second aspect of the invention provides a method for constructing a recombinant plasmid of hKLK1, which is obtained by connecting the hKLK1 gene to a classical expression clone pEZ-Lv 105.

Preferably, the method for constructing the hKLK1 recombinant plasmid comprises the following steps in sequence:

(1) designing a primer for PCR amplification by taking the hKLK1 gene sequence as a template, wherein the primer is as follows:

the sequence of the upstream primer is as follows: 5'-GCGGTAGGCGTGTACGGT-3'

The sequence of the downstream primer is as follows: 5'-ATTGTGGATGAATACTGCC-3', respectively;

(2) carrying out electrophoresis on the PCR amplification product obtained in the step (1), then carrying out gel cutting recovery and purification, and carrying out enzyme cutting by using restriction enzymes BstBI and XhoI so that the two ends of the obtained hKLK1 gene sequence have BstBI and XhoI enzyme cutting sites;

(3) ligating the DNA fragment obtained in the step (2) to an pEZ-Lv105 vector;

(4) screening, recombining, cloning and enzyme digestion identification by using a PCR method.

Wherein, the PCR amplification reaction system in the step (1) comprises:

5×Buffer，5μl；

10mM dNTP，0.5μl；

20mM Mg²⁺，2.5μl；

template, 1. mu.l;

5pmol/L of upstream primer mfat-1-F, 0.5. mu.l;

5pmol/L downstream primer mfat-1-R, 0.5. mu.l;

5U/μl UltraPF DNA Polymerase，0.2μl；

deionized water was added to 25. mu.l.

The PCR amplification reaction procedure in the step (1) is as follows: pre-denaturing at 98 ℃ for 1min, denaturing at 98 ℃ for 10sec, annealing to 5 ℃ for 30sec, and extending at 72 ℃ at a rate of 1-4Kb/min for 7min for 25 cycles; finally, the mixture is stored at 4 ℃.

The reaction system for screening the recombinant clone by the PCR method in the step (2) comprises:

10×Taq Reaction Buffer(Mg plus)，2.5μl；

10mM dNTP，0.5μl；

5U/μl Taq，0.2μl；

deionized water, 4.8. mu.l.

The PCR amplification reaction procedure in the step (2) is as follows: pre-denaturing at 94 ℃ for 5min, denaturing at 94 ℃ for 30sec, annealing to 58 ℃ for 30sec, and extending at 72 ℃ at a rate of 1Kb/min for 7min for 25 cycles; finally, the mixture is stored at 4 ℃.

The third aspect of the invention provides a lentivirus particle with high expression of hKLK1, which is obtained by connecting the recombinant plasmid of hKLK1 or the recombinant plasmid constructed by the method for constructing the recombinant plasmid of hKLK1 into lentivirus. Preferably, the administration mode is one-time intravenous injection and is based on HIV framework slow virus packaging plasmid Lenti-Pac^TM。

The fourth aspect of the invention provides a method for constructing a lentivirus particle with high expression of hKLK1, which comprises the following steps in sequence:

(1) Lenti-Pac lentivirus packaging plasmid^TMPackaging the hKLK1 recombinant plasmid;

(2) and (4) measuring the titer.

Preferably, the step (1) adopts EndoFectin^TMLenti and TiterBoost^TMTransfection reagent hKLK1 recombinant plasmid and lentivirus packaging plasmid Lenti-Pac^TMTransfection into 293Ta cells for co-transfection, more preferably, step (1) comprises the steps of:

culturing packaging cells

② preparation of DNA-EndoFectin transfection Complex

③ transfection of packaging cells

And fourthly, harvesting the lentivirus particles.

The fifth aspect of the invention provides an application of the hKLK1 recombinant plasmid or the hKLK1 recombinant plasmid constructed by the method for constructing the hKLK1 recombinant plasmid, or the lentiviral particle with high expression of hKLK1 or the lentiviral particle with high expression of hKLK1 constructed by the method for constructing the lentiviral particle with high expression of hKLK1 in preparing a medicament for treating viral myocarditis and myocardial fibrosis.

The sixth aspect of the invention provides a medicament, which comprises the hKLK1 recombinant plasmid or the hKLK1 recombinant plasmid constructed by the method for constructing the hKLK1 recombinant plasmid, or the high-expression hKLK1 lentiviral particle constructed by the method for constructing the lentiviral particles with high expression hKLK1 or the lentiviral particles with high expression hKLK1, and a medicament vector. Wherein the drug carrier is acceptable in drug preparation. The medicament can be prepared into clinically acceptable dosage forms by preparing the hKLK1 recombinant plasmid or the hKLK1 recombinant plasmid virus particles, a medicament carrier and conventional auxiliary materials according to a conventional process.

Human tissue kallikrein 1 (hKLK 1) is a serine protein kinase and belongs to one of the active ingredients of the kallikrein-kinin system (KKS). The invention adopts a gene therapy method, utilizes the virus particles to bring the hKLK1 gene into a VMC mouse body, compared with a control group mouse, the collagen fibers proliferated in a mouse myocardial interstitial matrix are obviously reduced, the proliferation of the myocardial collagen fibers is obviously light, the ratio of I/III collagen is obviously reduced, the expression of transforming growth factor beta1 (TGF-beta 1) in the myocardium is obviously reduced, and the hKLK1 can play the roles of inhibiting the VMC myocardial fibrosis and improving the cardiac function by reducing the expression of the TGF-beta 1, thereby having the application prospect of being developed into a medicament. Because of the lack of serine protein kinase in mammals, it is necessary to introduce the hKLK1 gene into the body using a lentiviral vector or an adeno-associated viral vector.

Compared with the prior art, the hKLK1 recombinant plasmid (pEZ-Lv105-hKLK1) can be used for preparing the medicine for treating viral myocarditis and myocardial fibrosis. The lentivirus is used as a vector, and the gene recombination technology is utilized to construct the lentivirus particle (EZ. hKLK1) of the high expression hKLK1, so that the lentivirus particle is a safe and nontoxic medicine capable of inhibiting VMC myocardial fibrosis, can meet the use requirement, and has the advantages of simple construction method, easy operation and good application prospect.

Drawings

FIG. 1 is an electrophoretogram of PCR products of example 1.

FIG. 2 is an electrophoretogram of PCR products of example 1.

FIG. 3 shows the restriction identification electrophoretogram of plasmid BsrGI and AflII of pEZ-Lv105-hKLK1 from example 1.

FIG. 4 is a graph of body weight ratios of the hearts of each group of example 3.

FIG. 5 is a micrograph of the myocardial tissue morphology of each group of mice of example 3.

FIG. 6 is a graph showing the expression of type I and type III collagens in each of the myocardium of example 3.

FIG. 7 is a graph showing the expression of myocardial TGF-. beta.1 proteins of each group in example 3.

Detailed Description

To better illustrate the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to specific examples. It should be noted that the following implementation of the method is a further explanation of the present invention, and should not be taken as a limitation of the present invention.

Example 1: the gene sequence of the hKLK1 recombinant plasmid (pEZ-Lv105-hKLK1) is shown as SEQ ID:1, and the specific process is as follows:

(1) PCR amplification

Taking the gene sequence of hKLK1 as a template, designing primers for PCR amplification to ensure that the two ends of the obtained gene sequence of hKLK1 have BstBI and XhoI enzyme cutting sites, wherein the primers are as follows:

the sequence of the upstream primer is as follows: 5'-GCGGTAGGCGTGTACGGT-3'

the PCR reaction system is shown in Table 1.

TABLE 1PCR reaction System

The PCR amplification reaction program is as follows: pre-denaturing at 98 ℃ for 1min, denaturing at 98 ℃ for 10sec, annealing to 5 ℃ for 30sec, and extending at 72 ℃ at a rate of 1-4Kb/min for 7min for 25 cycles; finally, the mixture is stored at 4 ℃.

The PCR product was identified by electrophoresis, and the results are shown in FIG. 1, in which Lane 1: marker 6000; lane 2: hKLK1(800bp), the size of the DNA fragment was estimated against marker, and positive clones containing the desired fragment were selected. Using OMEGA in combination

The Gel Extraction Kit Gel was used to recover and purify the PCR product.

(2) Enzyme digestion

The restriction enzymes BstBI and XhoI were used for digestion, and the digestion system is shown in Table 2.

TABLE 2 enzyme digestion System

Placing the reaction tube into a constant-temperature water bath kettle at 37 ℃ for incubation for more than 3h, taking out the enzyme digestion product, placing the enzyme digestion product into a water bath kettle at 80 ℃ for incubation for 20min, and inactivating the restriction enzyme; by OMEGA

Recovering the enzyme digestion product by using Gel Extraction Kit.

(3) Connection of

The target DNA fragment and pEZ-Lv105 vector are subjected to Fast-fusion TM reaction to obtain Fast-fusion TMCLONAse. Cloning reactions were prepared on ice as shown in table 3. mu.L of Fast-fusion (TM) clone was added to the well mixed reaction solution, the reaction tube wall was flicked to disperse the solution evenly, incubated at 25 ℃ for 15min and then stored on ice.

TABLE 3 reaction solution formulation

(4) Culturing and transforming:

1. the competent cells were removed from the-80 ℃ freezer and thawed on ice.

2. The sterilized 1.5ml centrifuge tubes were removed from the clean bench and their caps were marked with the appropriate numbers using a marker pen.

3. Mu.l of the ligation was removed using a pipette gun to the bottom of a labeled 1.5ml centrifuge tube and placed in ice.

4. The thawed competent cells were mixed well in a clean bench by pipette, 50. mu.l was added to the bottom of the 1.5ml centrifuge tube in step 3 and ice-cooled for 30min, at which time the water bath was set at 42 ℃.

5. Standing in 42 deg.C water bath, thermally shocking for 45sec, and rapidly transferring to ice box for 2-4 min.

6. The alcohol lamp was ignited on a clean bench, 400. mu.l of S.O.C medium was added by using a pipette, and then the mixture was incubated for 1 hour at 37 ℃ on a shaker at 180 rpm.

7. Taking out the converted substance from the shaking table, placing 200 μ l in an ultraclean workbench, taking 200 μ l with a pipette, spreading the converted substance uniformly, drying, placing the LB plate upside down in a 37 deg.C constant temperature incubator, and culturing for 12-16 h.

(5) Screening of recombinant clones by PCR

1. The LB plate was inverted, and the single clones on the plate were randomly selected and numbered at room temperature.

2. Each PCR tube was filled with 16. mu.l of ddH2O and 1. mu.l (5 pmol/. mu.l) of forward and reverse vector primers, and the tip was gently spotted on an LB plate, and then the tip stained with the cells was placed in the corresponding PCR tube and blown down several times.

3. The other components of the PCR were dispensed as PCR mix into each PCR tube to a total volume of 25. mu.l, then mixed well and the tube was capped, the PCR mix formulation is shown in Table 4.

TABLE 4PCR mix formulation

4. And (3) centrifuging the PCR mixture mixed with the thalli for a short time, and then placing the mixture into a PCR instrument, wherein the PCR reaction procedure is as follows: pre-denaturing at 94 ℃ for 5min, denaturing at 94 ℃ for 30sec, annealing to 58 ℃ for 30sec, and extending at 72 ℃ at a rate of 1Kb/min for 7min for 25 cycles; finally, the mixture is stored at 4 ℃.

5. The PCR product was detected by electrophoresis, and the result is shown in FIG. 2, and the size of the DNA fragment was estimated against marker, and a positive clone containing the desired fragment was selected. And wherein Lane1 is Marker 6000; lane2 is colony 1 PCR; lane3 is colony 2 PCR; lane4 is colony 3 PCR; lane5 is colony 4 PCR; lane6 is colony 5 PCR; lane6 is colony 5 PCR; lane7 was colony 6 PCR.

6. Selecting positive clones, inoculating the positive clones into 5ml LB/liquid culture medium containing antibiotics, and performing shake culture at 37 ℃ for 12-16h to prepare for extracting plasmids.

7. By OMEGA

Plasmid Mini Kit I extracts plasmid DNA.

(6) Enzyme digestion identification

The restriction enzyme BsrGI was used, and the restriction system is shown in Table 5.

TABLE 5 enzyme digestion System

And (3) putting the reaction tube into a constant-temperature water bath kettle at 37 ℃ for incubation for 3h, taking out the reaction tube, putting the reaction tube into a water bath kettle at 65 ℃ or 80 ℃ for incubation for 20min, and inactivating the restriction enzyme. 0.5. mu.l of plasmid which has not been digested and 5. mu.l of the digestion product are respectively taken for electrophoresis to detect whether the digestion is complete, as shown in FIG. 3, which shows that the plasmid digestion is complete. Wherein Lane1 is Marker 6000, Lane2 is plasma ofpEZ-Lv105-hKLK1-01, Lane3 is plasma of pEZ-Lv105-hKLK1-01cut by BsrGI, Lane4 is plasma of pEZ-Lv105-hKLK1-01cut by AflII, Lane5 is Marker 3000, and Lane6 is Marker 15000. Placing the reaction tube in a 37 deg.C constant temperature water bath for over 3h, taking out enzyme digestion product, placing in 80 deg.C water bath, incubating for 20min to inactivate restriction enzyme, and treating with OMEGA

Recovering the enzyme digestion product by using Gel Extraction Kit.

(7) Sequencing identification

The plasmid enzyme digestion is correct, and the result is consistent with Genbank after sequencing verification. The sequencing result is correct, and the small/medium extraction kit of OMEGA endotoxin-free plasmid is used for extracting the endotoxin-free plasmid.

Example 2: construction of Lentiviral particles highly expressing hKLK1

Packaging of plasmid Lenti-Pac by lentivirus^TMThe viral particles obtained after packaging the hKLK1 recombinant plasmid are based on HIV framework and are a lentivirus packaging plasmid Lenti-Pac^TMThe construction method comprises the following steps:

(1) culturing packaging cells

The amount of the DNA was about 1.3 × 10 two days before transfection⁶-1.5×10⁶The 293Ta cells were inoculated into 10cm plates, and 10mL of 10% heat-inactivated fetal bovine serum was addedDMEM medium at 5% CO₂Cells were plated and cultured at 37 ℃. For example, 293Ta cells reach 70-80% confluence on the day of transfection, and optimal packaging is relatively easy to achieve.

(2) Preparation of DNA-EndoFectin transfection Complex

To a sterile EP tube (A tube), 2.5. mu.g of hKLK1 recombinant plasmid and LentiPac were added^TMThe packaged plasmids were mixed at 5.0. mu.L (0.5. mu.g/. mu.L), and Opti-MEMI was added to make up to 200. mu.L (at this time, the total amount of plasmid in tube A was 5. mu.g). Another sterile EP tube (B tube) was added with 15. mu.L of EndoFectin Lenti transfection reagent and supplemented to 200. mu.L with Opti-MEMI. While gently swirling tube A, the solution from tube B was added dropwise to tube A (note: the order of addition was not reversed) until the addition of the solution from tube B was complete. And after the solutions are mixed, standing at room temperature for 10-25min to complete the preparation of the DNA-EndoFectin transfection compound.

(3) Transfection of packaging cells

On the day of transfection operation, taking out the cells plated and cultured in the step 1 from the incubator, and uniformly dropwise adding the transfection compound prepared in the step 2 to the cell plate; the act of sucking up the transfection complex needs to be gentle and does not repeatedly blow up the transfection complex. After the addition, the cell plate was shaken horizontally in a 8-shape (gentle motion was required) to disperse the transfection complex uniformly. After completion of transfection, at 5% CO₂Culturing at 37 deg.C for 8-14 h; after 8-14h of the transfection procedure, the old medium containing the transfection complex is removed and fresh DMEM medium containing 2-5% heat-inactivated fetal calf serum, penicillin and streptomycin is added (e.g., 10mL DMEM is added to a 10cm plate). At this step, 1/500 volumes of TiterBoost may be added to fresh DMEM medium^TMTiter enhancers (e.g., 20. mu.L TiterBoost added to 10cm plates) and continued at 5% CO₂And cultured at 37 ℃ to prepare for harvesting the lentiviral particles.

(4) Harvesting Lentiviral particles

Cell culture fluid is collected after 48h of transfection operation, and cell debris is removed by centrifugation at 2000 Xg for 10min, so as to obtain crude virus fluid of lentivirus.

(5) Titre determination

Chronic diseaseThe lentivirus titer obtained was determined by the virus infection titer (TCID50) method and calculated (calculation formula: 1TU ═ 100 copies of viral gene RNA), and the result showed that the virus titer was 2.4 × 10⁸TU/ml。

Example 3: effect of Lentiviral particles (EZ. hKLK1) highly expressing hKLK1 on treating myocardial fibrosis in viral myocarditis

(1) Establishment of VMC mouse model

1. 125ml of pEZ-Lv105-hKLK1 was injected into VMC mice by tail vein injection, and the empty vector group (EZ. null) mice were injected with 125ul of empty vector (lentiviral particles incapable of expressing hKLK1) by tail vein injection, and the remaining groups (Model group, normal group (Sham)) of mice were injected with 125ul of physiological saline by tail vein injection, and the number of mice in each group was 6.

After 2.30 days, the cardiac function of the mice was examined by cardiac ultrasound.

3. Mice were sacrificed and myocardial tissue was preserved and tested for heart/body weight ratio, myocardial collagen content, myocardial collagen levels type i and type iii, and myocardial transforming growth factor beta1 (TGF-beta 1) protein expression.

(2) Results and discussion

1. Body weight ratio of heart

As shown in FIG. 4, the heart-to-weight ratio was increased in the Model group compared to the Sham group, with statistical significance of the difference (P < 0.05). Compared with the Model group, the heart weight ratio of the hKLK1 administration group (EZ. hKLK1) is reduced, the difference is statistically significant (P is less than 0.05), and the EZ.null group difference is not statistically significant (P is more than 0.05).

2. Cardiac ultrasound

As shown in Table 6, Model group mice showed an increase in both the left ventricular end-diastolic diameter (LVEDd) and the left ventricular end-systolic diameter (LVEDs) compared to the Sham group, and the calculated left ventricular Fractional Shortening (FS) and Ejection Fraction (EF) were decreased, with statistical differences (P < 0.05). Compared with the Model group, the EZ.hKLK1 group has reduced LVEDd and LVEDs, increased FS and EF, and statistically significant difference (P is less than 0.05), while the EZ.null group has no statistically significant difference (P is more than 0.05).

TABLE 6 levels of LVEDd, LVEDs, FS and EF for each group of mice

3. Myocardial collagen content determination

As shown in Table 7, the Model group showed statistically significant differences in myocardial collagen content (P < 0.05) compared to the Sham group. Compared with the Model group, the content of myocardial collagen of the EZ.hKLK1 group is obviously reduced, the difference has statistical significance (P is less than 0.05), and the content of myocardial collagen of the EZ.null group has no statistical significance (P is more than 0.05).

TABLE 7 myocardial collagen content in each group

4. Histopathological characterization of myocardium

As shown in FIG. 5, the structure of the myocardial cells of the Sham group mice is clear, the muscle fibers are arranged regularly and are connected completely without any inflammatory infiltration, necrosis and fibrosis. The Model group mice can see obvious myocardial fibrosis, part of the mice are changed like glass, the fibrotic foci are connected with cardiac muscle, and a small amount of lymphocyte infiltration can be seen. Null was essentially the same as Model group, and interstitial hyperplastic collagen fibers were significantly reduced in the ez. hklk1 group.

5. Expression of type I and type III collagen

As shown in fig. 6, after staining with sirius red, Sham group mice were shown, with a small amount of red, delicate collagen visible in the myocardial intercellular spaces; heart interstitium of a Model group mouse can see type I collagen and type III collagen, and the I collagen with large hyperplasia is overlapped repeatedly, arranged disorderly, has uneven diameter and has stronger birefringent light; the proliferation of myocardial collagen fibers of the EZ.hKLK1 group is obviously lighter than that of the Model group, and the trend of the proliferation is reduced; the ez.null mice had insignificant changes in myocardial collagen fibril proliferation compared to the Model group. The I/III collagen ratios in the Model group were significantly higher and the differences were statistically significant (P < 0.05) compared to the Model group, the I/III collagen ratios in the EZ.hKLK1 group were significantly lower and the differences were statistically significant (P < 0.05) compared to the Model group, while the I/III collagen ratios in the EZ.null group were not significantly changed and the differences were not statistically significant (P > 0.05), as shown in Table 8.

TABLE 8 collagen I/III ratio of myocardium in groups of mice

6. Expression of myocardial TGF-beta 1

As shown in FIG. 7, the expression of TGF-. beta.1 was significantly increased in the Model group compared to the Sham group, and the difference was statistically significant (P < 0.05). Compared with the Model group, the expression of TGF-beta 1 in the EZ.null group has no significant difference (P is more than 0.05), while the expression of TGF-beta 1 in the EZ.hKLK1 group is significantly reduced, and the difference has statistical significance (P is less than 0.05). In the research, the hKLK1 can play the roles of inhibiting VMC myocardial fibrosis and improving cardiac function by down-regulating the expression of TGF-beta 1, and has an application prospect of being developed into a medicament.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Sequence listing

<110> Songshan lake Central Hospital, Dongguan City (Shilong people Hospital, Dongguan city, third people Hospital, Dongguan city, cardiovascular disease institute)

<120> hKLK1 recombinant plasmid, high-expression hKLK1 lentiviral particle, and construction method and application thereof

<141>2020-05-19

<160>1

<170>SIPOSequenceListing 1.0

<210>2

<211>863

<212>DNA

<213>Artificial Sequence

<400>2

catggccaat atgattttat tttgactgat agtgacctgt tcgttgcaac aaattgatga 60

gcaatgcttt tttataatgc caactttgta caaaaaagca ggcttggaag gagttcgaac 120

catgtggttc ctggttctgt gcctcgccct gtccctgggg gggactggtg ctgcgccccc 180

gattcagtcc cggattgtgg gaggctggga gtgtgagcag cattcccagc cctggcaggc 240

ggctctgtac catttcagca ctttccagtg tgggggcatc ctggtgcacc gccagtgggt 300

gctcacagct gctcattgca tcagcgacaa ttaccagctc tggctgggtc gccacaactt 360

gtttgacgac gaaaacacag cccagtttgt tcatgtcagt gagagcttcc cacaccctgg 420

cttcaacatg agcctcctgg agaaccacac ccgccaagca gacgaggact acagccacga 480

cctcatgctg ctccgcctga cagagcctgc tgataccatc acagacgctg tgaaggtcgt 540

ggagttgccc accgaggaac ccgaagtggg gagcacctgt ttggcttccg gctggggcag 600

catcgaacca gagaatttct catttccaga tgatctccag tgtgtggacc tcaaaatcct 660

gcctaatgat gagtgcaaaa aagcccacgt ccagaaggtg acagacttca tgctgtgtgt 720

cggacacctg gaaggtggca aagacacctg tgtgggtgat tcagggggcc cgctgatgtg 780

tgatggtgtg ctccaatgtg tcacatcatg gggctacctc ccttgtggca cccccaataa 840

gccttctgtc gccttcaaag tgc 863

Claims

1. The hKLK1 recombinant plasmid is characterized in that the hKLK1 gene is cloned, and the gene sequence is shown as SEQ ID: 1.

2. A method for constructing the hKLK1 recombinant plasmid according to claim 1, wherein the hKLK1 gene is ligated to a classical expression clone pEZ-Lv 105.

3. The process for constructing recombinant hKLK1 plasmid according to claim 2, which comprises the following steps in sequence:

the sequence of the upstream primer is as follows: 5'-GCGGTAGGCGTGTACGGT-3'

(3) ligating the DNA fragment obtained in the step (2) to an pEZ-Lv105 vector;

4. A lentiviral particle highly expressing hKLK1, wherein the lentiviral particle is obtained by connecting the hKLK1 recombinant plasmid of claim 1 or the recombinant plasmid constructed by the method for constructing the hKLK1 recombinant plasmid of claim 2 or 3 to a lentivirus.

5. The lentiviral particle with high expression of hKLK1 according to claim 4, wherein the administration mode is intravenous injection.

6. The lentiviral particle with high expression of hKLK1 according to claim 4, which is a lentiviral packaging plasmid Lenti-Pac based on HIV framework^TM。

7. The method for constructing the lentiviral particle highly expressing hKLK1 according to any one of claims 4 to 6, wherein the method comprises the following steps in sequence:

(2) and (4) measuring the titer.

8. The method for constructing high-expression hKLK1 lentivirus particles according to claim 7, wherein the method comprisesThe step (1) adopts EndoFectin^TMLenti and TiterBoost^TMTransfection reagent hKLK1 recombinant plasmid and lentivirus packaging plasmid Lenti-Pac^TMTransfection into 293Ta cells for co-transfection.

9. The use of the hKLK1 recombinant plasmid according to claim 1, or the hKLK1 recombinant plasmid constructed by the method for constructing the hKLK1 recombinant plasmid according to claim 2 or 3, or the lentiviral particle with high expression of hKLK1 according to any one of claims 4 to 6, or the lentiviral particle with high expression of hKLK1 constructed by the method for constructing the lentiviral particle with high expression of hKLK1 according to claim 7 or 8 in the preparation of a medicament for treating viral myocarditis myocardial fibrosis.

10. A medicament, comprising the hKLK1 recombinant plasmid of claim 1, or the hKLK1 recombinant plasmid constructed by the method for constructing the hKLK1 recombinant plasmid of claim 2 or 3, or the lentiviral particle with high expression of hKLK1 of any one of claims 4 to 6, or the lentiviral particle with high expression of hKLK1 constructed by the method for constructing the lentiviral particle with high expression of hKLK1 of claim 7 or 8, and a pharmaceutical carrier.