Preparation of PTC stable cell line by using inhibitory tRNA system and application thereof
阅读说明：本技术 利用抑制性tRNA系统制备PTC稳定细胞系及应用 (Preparation of PTC stable cell line by using inhibitory tRNA system and application thereof ) 是由 夏青 王宇 杨琦 牛振岚 于 2018-08-20 设计创作，主要内容包括：本发明属于生物制药领域,具体涉及抑制性tRNA调控提前终止密码子(PTC)通读的稳定细胞系制备方法及应用。本发明主要涉及构筑20种对应三种终止密码子的抑制性tRNA,利用20种抑制性tRNA通读提前终止密码子(PTC,UAG/UAA/UGA)。本发明还涉及串联多拷贝正交tRNA的质粒载体的构建方法,以及借助线性化质粒、慢病毒或Tol2转座子系统将串联的抑制性tRNA基因稳定整合到细胞基因组的方法。本发明进一步涉及稳定细胞系的应用,如包装复制缺陷型(PTC)病毒疫苗。(The invention belongs to the field of biological pharmacy, and particularly relates to a preparation method and application of a stable cell line for regulating and controlling early termination codon (PTC) readthrough by using suppressor tRNA. The invention mainly relates to the construction of 20 kinds of suppressive tRNA corresponding to three kinds of stop codons, and the 20 kinds of suppressive tRNA are used for reading early stop codons (PTC, UAG/UAA/UGA). The invention also relates to methods for constructing plasmid vectors with multiple copies of orthogonal tRNAs in tandem, and methods for stably integrating suppressor tRNA genes in tandem into the genome of a cell using linearized plasmids, lentiviruses, or a Tol2 transposable subsystem. The invention further relates to the use of stable cell lines, such as packaging replication-defective (PTC) virus vaccines.)
1. An engineered suppressor tRNA (stRNA), wherein the stRNA is derived from a eukaryote, wherein the stRNA recognizes a premature stop codon, and wherein the stRNA is capable of carrying a natural amino acid.
2. The stRNA according to claim 1, wherein the gene of the stRNA is selected from the group consisting of the sequences represented by SEQ ID NOS 1-20.
3. A translation system comprising an aminoacyl tRNA synthetase and an stRNA according to claim 1 or 2, wherein the genes for the aminoacyl tRNA synthetase and the stRNA are located on the same vector.
4. The translation system of claim 3, further comprising a eukaryotic cell; in one embodiment, the eukaryotic cell is selected from 293T, BHK-21, MDCK, RD, Vero or CHO cells.
5. A eukaryotic cell comprising an aminoacyl tRNA synthetase and an stRNA according to claim 1 or 2, wherein genes for the aminoacyl tRNA synthetase and the stRNA are introduced into the eukaryotic cell in the same vector; in one embodiment, the eukaryotic cell is selected from 293T, BHK-21, MDCK, RD, Vero or CHO cells.
6. A method of making the eukaryotic cell of claim 5, comprising:
(1) respectively connecting 318 single-copy inhibitory tRNAs to the carriers, detecting the read-through efficiency of the inhibitory tRNAs, and determining the inhibitory tRNAs with the highest read-through efficiency corresponding to the 20 amino acids;
(2) respectively designing 20 multi-copy tandem inhibitory tRNA fragments according to the sequences of the 20 inhibitory tRNA determined in the step (1), and obtaining 20 multi-copy tandem inhibitory tRNA vectors which simultaneously contain a reporter gene, a resistance gene and high-reading efficiency by using the fragments and mutated fluorescent protein;
(3) and (3) transducing the suppressor tRNA vector in (2) into eukaryotic cells, screening with antibiotic, selecting the fluorescent monoclone, expanding and culturing to obtain stable cell line and stable cell line with integrated mutant green fluorescent protein reporter gene and multicopy stRNA.
7. A method of preparing a vaccine comprising a replication defective (PTC) virus using the eukaryotic cell of claim 5, comprising the steps of:
(1) selecting an amino acid site of a desired mutation in the amino acid sequence of a viral protein of interest;
(2) mutating the codon of the amino acid at the selected position in the step (1) into a stop codon UAG, UAA or UGA in the nucleic acid molecule for encoding the target protein in the step (1);
(3) operably linking the mutated nucleic acid obtained in (2) with a suitable vector to obtain an expression vector for the nucleic acid;
(4) transfecting the eukaryotic cell of claim 5 with the expression vector of the mutated nucleic acid obtained in (3), culturing the host cell after successful transfection in a culture medium, and collecting the virus at an appropriate time;
(5) the packaging titer and activity of the virus were tested.
8. A virus containing site-directed mutations produced using the method of claim 7.
9. A composition comprising an effective amount of the site-directed mutant virus of claim 8.
10. A vaccine comprising an effective amount of the site-directed mutant virus of claim 8.
11. A pharmaceutical composition comprising an effective amount of the site-directed mutant virus of claim 8, and a pharmaceutically acceptable excipient.
12. Use of the site-directed mutant virus of claim 8 in the preparation of an attenuated live vaccine, and in the preparation of a medicament for the prevention and treatment of viral infection.
13. Use of the site-directed mutant virus of claim 8 in the prevention and treatment of infection.
Technical Field
The invention belongs to the field of biological pharmacy, and particularly relates to a preparation method and application of a stable cell line for regulating and controlling read-through of advanced termination codons (PTC) by using suppressor tRNA. The invention mainly relates to the construction of 20 kinds of suppressive tRNA corresponding to three kinds of stop codons, and the 20 kinds of suppressive tRNA are used for reading early stop codons (PTC, UAG/UAA/UGA). The invention also relates to methods for constructing plasmid vectors with multiple copies of orthogonal tRNAs in tandem, and methods for stably integrating suppressor tRNA genes in tandem into the genome of a cell using linearized plasmids, lentiviruses, or a Tol2 transposable subsystem. The invention further relates to the use of stable cell lines, such as packaging replication-defective (PTC) virus vaccines.
Background
Suppressive tRNA read-through nonsense mutation
The 61 codons in the human genome can be recognized by tRNA, encode 20 amino acids, and the three stop codons (UAG, UAA, UGA) do not have corresponding tRNA recognition, do not encode amino acids, and then terminate translation. However, it has been found that, in the presence of tRNA which recognizes the stop codon, the stop codon encodes an amino acid, and that protein translation proceeds normally. The tRNA capable of recognizing the stop codon is nonsense mutation suppressive tRNA. Inhibitory tRNAs exist in a wide variety of ways, and are found in both plant and animal cells. However, inhibitory tRNAs are not easily detected because they are present in very small amounts in cells.
The nonsense mutation suppressor tRNA is generated by mutation of the anticodon loop base of the normal coding amino acid tRNA, the mutated tRNA can recognize a stop codon and is completely complementary and paired with the stop codon, meanwhile, the mutated tRNA can still carry natural amino acid, specific amino acid can be inserted into the position of the early stop codon, and the nonsense mutation can be read through. Based on the characteristic that the suppressor tRNA can read through nonsense mutation, the document reports that the suppressor tRNA is used for reading through protein containing a premature stop codon in prokaryotic cells and eukaryotic cells to recover protein expression. In addition, although suppressor trnas were able to restore expression of proteins containing nonsense mutations, there were large differences in read-through efficiencies between suppressor trnas. Therefore, it is necessary to find efficient suppressor tRNAs, and it is important to construct various cell lines stably expressing suppressor tRNAs with efficient read-through stop codons, especially to compare the difference of read-through efficiency in mammalian cells.
PTC technology and its application bottleNeck
Through years of research, people have more comprehensive understanding on the translation mechanism of prokaryotic ribosome, the crystal and electron microscope structures of different functional states of various ribosomes are analyzed, and the structures of most aminoacyl tRNA synthetases are obtained. Based on these findings, a technique of genetic codon expansion, which introduces amber stop codon (TAG) into genome, encodes various unnatural amino acids using an exogenous unnatural amino acid bioorthogonal translation system, and inserts them at a site in living organisms, has been developed in recent years. To date, this technique has successfully targeted expression of several unnatural amino acids, including affinity tags and photoisomerized, carbonyl amino acids, and glycosylated amino acids, among proteins in living cells, conferring novel physical, Chemical, and physiological properties to these proteins (L.Wang et al, 2001, Science 292: 498-500; J.W.Chin et al, 2002, Journal of the American Chemical Society 124: 9026-9027; J.W.Chin, & P.G.Schultz, 2002, ChemBiochem 11: 1135-1137). The researches show that the technology can selectively introduce special chemical groups such as carbonyl, alkynyl, azide groups and the like into the protein, realize the site-specific modification of the protein and improve the property of the protein. Meanwhile, the technique can also be applied to the aspects of site-directed labeling, site-directed modification, control of replication, etc. of living organisms (such as viruses, bacteria, etc.) (Si L, etc., 2016, Science 354: 1170-1173).
The PTC technology is a virus vaccine development technology that introduces Premature Termination Codons (PTCs) into a virus genome to control replication and protein expression of viruses and make them dependent on exogenous unnatural amino acids. The PTC technology is really applied to the development of virus vaccines, and a problem to be solved urgently is how to construct engineering cells which are stably integrated and can express a large amount of orthogonal tRNA/aminoacyltRNA synthetase/GFP reporter genes. The construction of the engineering cell for stably integrating the orthogonal tRNA/aminoacyl tRNA synthetase/GFP reporter gene is realized, and the application of the PTC technology in the development of virus vaccines is greatly promoted. However, the current construction technology of the engineering cell still has the following difficulties:
firstly, because the transcription and processing of tRNA are different from protein, how to realize the efficient and stable expression of orthogonal prokaryotic tRNA in eukaryotic cells is still an international problem; secondly, according to the traditional method, engineering cells simultaneously and stably expressing three different exogenous gene elements need to be subjected to three rounds of gene transfection or virus transduction and corresponding screening processes of three different antibiotics, and because the cells are poor in state and difficult to survive under the pressure of the multiple antibiotics, and the used antibiotics are expensive to screen at the same time, the cell line construction process is complicated, the success rate is low, and the cost is high; the cell source of the engineering cell which is successfully constructed at present and stably integrates the orthogonal tRNA/aminoacyltRNA synthetase/GFP reporter gene is human embryonic kidney HEK293T cell, the cell has stronger adaptability, but the application in the rescue and development process of virus vaccine is limited to AIDS virus and influenza virus, the rescue efficiency of most viruses in the cell line is lower, and the wide application is difficult to obtain; in addition, the conventional gene codon expansion technology is mostly limited to amber stop codon (TAG), which is determined based on the lowest usage frequency of amber stop codon (TAG) in the escherichia coli model for studying gene codon expansion technology, but studies have shown that the codon usage frequency of some viral genes is not completely matched with that of host cells, which is another limitation of the PTC technology applied to the development of viral vaccines.
Therefore, a plurality of cell lines for stably expressing the inhibitory tRNA are constructed, endogenous natural amino acid is utilized to efficiently read the truncated protein, so that stable and efficient packaging of the PTC virus vaccine is realized, meanwhile, the safety problem caused by non-natural amino acid residues is solved, and the application of the PTC technology in the research and development of the virus vaccine is effectively promoted.
Application of Vero cells in vaccine production
In 1963, two scholars, y.yasumura and y.kawakita, at the university of thousand leaves of japan, developed a Vero cell line derived from renal epithelial cells of african green monkeys (Cercopithecus aethiops). In 1964, romizu, supplied the 93 rd generation Vero cells to the trimical virus laboratory (NIAID, NIH) in the uk. In 1979, the 113 th generation of Vero cells was supplied to the American Standard Culture Collection (ATCC) and passed to the 121 th generation to establish a cell bank. The Vero cell is a continuous cell line, can be continuously passaged in vitro, and is sensitive to various viruses, including SV-40, SV-5, measles virus, arbovirus, retrovirus, rubella virus, monkey virus, adenovirus, poliovirus, influenza virus, parainfluenza virus, respiratory syncytial virus, vaccinia virus and other various viruses. Therefore, Vero cells are widely applied to relevant biological detection in laboratories after being prepared, such as virus amplification, plaque detection and the like.
The Vero cells are rapidly developed for vaccine development since the nineties of the twentieth century and are approved by the world health organization and the national biological product regulation. In the last decade, the research of viral vaccines in China has been rapidly developed, and novel vaccines are continuously emerging. With the popularization and application of advanced cell culture technologies, such as bioreactor and fermenter cell suspension culture technologies, more production enterprises tend to select Vero cells for viral vaccine production.
Compared with primary cells, diploid cells and other subculture cell matrixes used for vaccine production, the Vero cells have the characteristics of convenience in ① source, capability of continuous subculture, high growth speed, sensitivity of ② to infection of various viruses, high virus multiplication titer, stable ③ genetic character, low malignant transformation degree, high biological safety, and non-harsh requirements on ④ culture conditions, and are easy to implement large-scale culture in a bioreactor.
Disclosure of Invention
Through thinking and research on the prior art, in order to improve the packaging efficiency of the virus and realize future industrial production, the inventor modifies all tRNA in the Transfer RNA database (http:// tRNA. biolin. uni-leipzing. de/DataOutput /), changes the tRNA anticodon loop to obtain inhibitory tRNA which is completely complementary and paired with the early stop codon, constructs the inhibitory tRNA by an SOE PCR method, and connects a 7sk promoter to the 5' end of the inhibitory tRNA to obtain 318 single-copy inhibitory tRNA carriers. Then, the read-through efficiency analysis is carried out in a GFP (green fluorescent protein) reporter gene model and a dual-luciferase reporter gene model to obtain 20 read-through efficienciesThe highest suppressor tRNA is
Advantages of the invention over other approaches may be realized in one or more of the following:
1. the obtained 20 kinds of inhibitory substances capable of efficiently reading through nonsense mutation
2. The construction of any multi-copy tandem suppressor tRNA which simultaneously contains a reporter gene and a resistance gene and has high reading efficiency is realized;
3. obtaining 20 stable cell lines carrying high-pass efficiency inhibitory tRNA;
4. by utilizing the stable cell line, specific natural amino acid can be efficiently inserted into any site of the target protein, so that the complete expression of the nonsense mutant protein, the efficient packaging of the PTC virus vaccine and the like are realized.
Specifically, in a specific embodiment of the invention, the high-reading-efficiency multicopy tandem suppressor tRNA is integrated in a host cell, Vero, mainly by the following steps: (1) constructing and detecting the read-through efficiency of 318 single-copy suppressive tRNA carriers through experiments, and determining 20 kinds of suppressive tRNA which respectively correspond to the amino acids and have the highest read-through efficiency; (2) designing, synthesizing and constructing 20 multi-copy tandem suppression tRNA carriers simultaneously containing a reporter gene, a resistance gene and high reading efficiency according to the sequences of the 20 suppression tRNA determined in the step (1); (3) and (3) electrotransfecting the Vero cell with the multi-copy series-connection suppressive tRNA carrier plasmid obtained in the step (2), adding 400 mu g/ml Hygromycin antibiotic for screening 48 hours after electrotransformation, selecting a monoclonal with green fluorescence, carrying out amplification culture, obtaining a stable cell line simultaneously integrating the multi-copy series-connection suppressive tRNA and a mutant green fluorescence protein reporter gene, and finally obtaining a stable cell line Vero-stRNA.
The principle of the suppressive tRNA read-through nonsense mutation is that: (1) in the normal translation process of cells, the premature stop codon is recognized by the first peptide chain releasing factor eRF1, while the normal tRNA cannot recognize the stop codon, eRF3 is a type of GTPase which depends on ribosome and the first peptide chain releasing factor, and eRFl is cooperated to promote the release of peptide chains from ribosome, and the translation process is stopped (Zhourevlova, G.et al. EMBO J,1995,14, 4065-72.). The constructed suppressor tRNA is obtained by modifying a normal tRNA through an anticodon loop, wherein the anticodon loop can be completely complementary and paired with stop codons UAG, UAA and UGA, and competes with eRF1 to recognize a premature stop codon, so that the tRNA with the anticodon loop can still carry corresponding amino acid, and therefore, the suppressor tRNA inserts the amino acid at the position of the premature stop codon, the translation process is continued, and nonsense mutation is read; (2) the 7sk promoter is connected to the end of the constructed inhibitory tRNA 5', so that the inhibition tRNA can be started to express in a large amount in mammalian cells, and the protein expression is finally recovered.
More specifically, the present invention provides:
1. 318 suppression tRNA expression vectors containing 7sk promoter sequences can realize the overexpression of any suppression TNRA in mammalian cells.
2.20 inhibitory Properties enabling efficient read-through of nonsense mutations
3.20 multicopy tandem suppressor tRNA carrier plasmids which simultaneously contain a reporter gene, a resistance gene and high read efficiency;
4. the stable cell line is Vero-Ast, Vero-Rst, Vero-Nst, Vero-Dst, Vero-Cst, Vero-Qst, Vero-Est, Vero-Gst, Vero-Hst, Vero-Ist, Vero-Lst, Vero-Kst, Vero-Mst, Vero-Fst, Vero-Pst, Vero-Sst, Vero-Tst, Vero-Wst, Vero-Yst and Vero-Vst, and is obtained by 1 round of plasmid stable electrotransfection and carries multiple copies of tandem inhibitory tRNA genes.
5. An influenza virus packaging plasmid system containing an early stop codon UAG and a multi-copy tandem suppression tRNA stable cell line with high read-through efficiency are used for efficiently packaging the PTC influenza virus vaccine.
In one aspect, the invention provides a cell line carrying an engineered human suppressor tRNA (stRNA) gene.
In one embodiment, wherein said stRNA is promoter-stRNA in multiple copy numbers.
In still another embodiment, wherein said stRNA is a6 copy number stRNA promoted by type-3Pol III promoter.
In one embodiment, wherein said stRNA is characterized in that the anticodon loop is CUA, UUA or UCA.
In still another embodiment, wherein said engineered human stRNA is represented by SEQ ID NOS: 1-20.
In one embodiment, the cell line is obtained by the steps of:
(1) respectively connecting 318 single copy inhibitory tRNA on a Bjmu carrier shown as SEQ ID NO. 24 and detecting the read-through efficiency thereof, and determining the inhibitory tRNA with the highest read-through efficiency corresponding to 20 amino acids;
(2) respectively designing 20 multi-copy tandem inhibitory tRNA fragments from the sequences of the 20 inhibitory tRNA determined in the step (1), and obtaining 20 multi-copy tandem inhibitory tRNA vectors pcDNA3.1-6 Gln-stRNA-GFP-Hygro which simultaneously contain a reporter gene, a resistance gene and high reading efficiency by using the fragments and mutant green fluorescent protein;
(3) the inhibitory tRNA vector pcDNA3.1-6 Gln-stRNA-GFP-Hygro in (2) is transduced into Vero cells, screened by hygromycin, and a monoclonal with green fluorescence is picked and is subjected to amplification culture to finally obtain a stable cell line, and a stable cell line integrating the mutant green fluorescent protein reporter gene and 6 copy numbers of stRNA is obtained;
in yet another embodiment, the stable cell line is Vero-Ast, Vero-Rst, Vero-Nst, Vero-Dst, Vero-Cst, Vero-Qst, Vero-Est, Vero-Gst, Vero-Hst, Vero-Ist, Vero-Lst, Vero-Kst, Vero-Mst, Vero-Fst, Vero-Pst, Vero-Sst, Vero-Tst, Vero-Wst, Vero-Yst, Vero-Vst.
In yet another aspect, the invention provides a method for producing a replication defective (PTC) virus vaccine comprising an unnatural amino acid using a cell line of the invention, comprising the steps of:
(1) selecting an amino acid site of a desired mutation in the amino acid sequence of a viral protein of interest;
(2) mutating the codon of the amino acid at the selected position in the step (1) into a stop codon UAG, UAA or UGA in the nucleic acid molecule for encoding the target protein in the step (1);
(3) operably linking the mutated nucleic acid obtained in (2) with a suitable vector to obtain an expression vector for the nucleic acid;
(4) transfecting the expression vector of the mutated nucleic acid obtained in the step (3) into the cell line of the invention, culturing the host cell after successful transfection in a culture medium, and collecting the virus at a proper time;
(5) the packaging titer and activity of the virus were tested.
In another aspect, the invention provides nucleic acid molecules encoding the muteins or peptides of the invention.
In one embodiment, the nucleic acid molecule of the mutant viral protein or peptide is characterized in that the codon encoding the unnatural amino acid is the stop codon UAG, UAA or UGA.
In yet another aspect, the invention provides viruses containing site-directed mutations made using the methods of the invention.
In another aspect, the invention provides compositions comprising an effective amount of a site-directed mutant virus of the invention.
In yet another aspect, the invention provides a vaccine comprising an effective amount of a site-directed mutant virus of the invention.
In another aspect, the present invention provides a pharmaceutical composition comprising an effective amount of the site-directed mutant virus of the present invention, and a pharmaceutically acceptable excipient.
In yet another aspect, the invention provides the use of the site-directed mutant virus of the invention in the preparation of an attenuated live vaccine, and in the preparation of a medicament for the prevention and treatment of viral infection.
In another aspect, the invention provides the use of the site-directed mutant viruses of the invention in the prevention and treatment of infection.
In one aspect, the invention provides an engineered suppressor trna (stRNA), wherein the stRNA is derived from a eukaryote, wherein the stRNA recognizes a premature stop codon, and wherein the stRNA is capable of carrying a natural amino acid.
In one embodiment, the gene of the stRNA is selected from the group consisting of the sequences shown in SEQ ID NOS: 1-20.
In another aspect, the invention provides a translation system comprising an aminoacyl-tRNA synthetase and an stRNA of the invention, wherein the genes for the aminoacyl-tRNA synthetase and the stRNA are located on the same vector.
In one embodiment, the translation system further comprises a eukaryotic cell; in one embodiment, the eukaryotic cell is selected from 293T, BHK-21, MDCK, RD, Vero or CHO cells.
In yet another aspect, the invention provides a method of making a eukaryotic cell of the invention.
In another aspect, the invention provides methods for producing a vaccine comprising a replication defective (PTC) virus using the eukaryotic cells of the invention.
Description of the drawings:
the following drawings are used to illustrate the advantageous effects of the present invention. It should be understood that these are for purposes of illustrating particular embodiments of the invention and are not intended to limit the scope of the invention.
FIG. 1: construction of Single copy suppressor tRNA and screening of read-through efficiency
A: the normal tRNA is used for modifying an anticodon ring to synthesize a suppressor tRNA;
b: the schematic diagram of the suppressive tRNA for reading the PTC is that the suppressive tRNA anticodon loop carrying the amino acid is completely complementary and paired with the early stop codon to read the PTC;
c: a Bjmu vector plasmid linked to a 7 sk-single copy suppressor tRNA;
d: read-through efficiency analysis in the GFP reporter gene model and the dual luciferase reporter gene model.
FIG. 2: construction of multi-copy tandem suppression tRNA carrier plasmid with high reading efficiency
A: multiple copies of tandem suppressor tRNA fragments;
b: cloning multiple copies of tandem suppressor tRNA fragments into pcDNA3.1/Hygro (+) vector with MfeI enzyme;
c: simultaneously carries a reporter gene, a resistance gene and a high-read-efficiency multi-copy tandem suppression tRNA carrier plasmid;
FIG. 3: procedure for screening Stable cell lines
Electrically transfecting Vero cells with the obtained multicopy tandem suppressor tRNA vector plasmid, adding 400 mu g/ml hygromycin antibiotic for screening 48 hours after the electric transfer, picking out a monoclonal with green fluorescence, continuously carrying out amplification culture with 200 mu g/ml hygromycin to obtain a stable cell line simultaneously integrating multicopy tandem suppressor tRNA and mutant green fluorescent protein reporter genes, and finally obtaining stable cell lines Vero-Ast, Vero-Rst, Vero-Nst, Vero-Dst, Vero-Cst, Vero-Qst, Vero-Est, Vero-Gst, Vero-Hst, Vero-Ist, Vero-Lst, Vero-Kst, Vero-Mst, Vero-Fst, Vero-Pst, Vero-Tst, Vero-Wst, Vero-Yst and Vero-Vst.
FIG. 4: identification of stable cell lines
A: tRNAPyl Gene copy number standard curve in Vero-PYL cells;
b: green fluorescent protein imaging of stable cell lines;
c: detecting the expression of the full-length green fluorescent protein of the stable cell line by western blot;
d: the stable cell line can efficiently save the PTC influenza virus vaccine in vitro.
Detailed Description
For the purposes of promoting an understanding of the invention, reference will now be made to certain embodiments and specific language will be used to describe the same. It should be understood, however, that these specific embodiments are not intended to limit the scope of the invention. Any alterations and further modifications in the described embodiments, and any further applications of the invention as described herein are contemplated as would normally occur to one skilled in the art to which the invention relates. All variations and embodiments which are equivalent to the present invention are included in the present invention.
Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
The media and assay conditions used in the examples of the present invention are those conventional in the art unless otherwise specified. The reagents used in the examples of the present invention were all commercially available unless otherwise specified.
In the following examples, the percentages are by mass unless otherwise specified.