CN1283630A - Trichosanthin mutant and its preparing process - Google Patents

Trichosanthin mutant and its preparing process Download PDF

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CN1283630A
CN1283630A CN 00119553 CN00119553A CN1283630A CN 1283630 A CN1283630 A CN 1283630A CN 00119553 CN00119553 CN 00119553 CN 00119553 A CN00119553 A CN 00119553A CN 1283630 A CN1283630 A CN 1283630A
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tcs
mtcs
mutant
amino
santhin
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柯一保
聂慧玲
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SHANGHAI INSTITUTE OF CELL BIOLOGY CAS
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SHANGHAI INSTITUTE OF CELL BIOLOGY CAS
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Priority to CN 00119553 priority Critical patent/CN1283630A/en
Priority to CNB011031026A priority patent/CN1152051C/en
Publication of CN1283630A publication Critical patent/CN1283630A/en
Priority to US09/905,247 priority patent/US7157552B2/en
Priority to EP01983407.6A priority patent/EP1307480B1/en
Priority to PCT/CN2001/001178 priority patent/WO2002012537A2/en
Priority to CNB018167012A priority patent/CN1208343C/en
Priority to AU2002214919A priority patent/AU2002214919A1/en
Priority to JP2002517821A priority patent/JP4418155B2/en
Priority to HK04104880A priority patent/HK1061872A1/en
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Abstract

A trichosanthin mutant is prepared through the mutational deformation of trichosanthin gene and using proper expression system. Its advantages include low sensibiligen nature, high bioactivity, high selectivity to target, and strong kill action to cancer cells, virus and HIV. It can also be used for metaphase induced labor or exfetation.

Description

A kind of MUTANT OF TRICHO-SANTHIN and preparation method
The present invention relates to genetically engineered, be specifically related to a kind of MUTANT OF TRICHO-SANTHIN (Mutantof Trichosanthin, MTCS) and the preparation method.
Snakegourd Root is the Chinese medicine of China's traditional medicine, derives from the root of cucurbitaceous plant snakegourd (Trichosanthes Kirilowii M.), and two over thousands of years clinical medicine applicating histories are arranged.Therefrom a kind of molecular weight of separation and purification is that the protein of 27KD is called Trichosanthin (Trichosanthin, TCS), its chemical nature is a kind of ribosome inactivating protein (Ribosome Inactivating Protein of strand, RIP), last the 4324th VITAMIN B4 of the special excision eukaryotic cell 28SrRNA of energy, thus blocking protein is synthetic.Has " clearing heat and detoxicating " effect.Sixties people further study the ability of " termination of pregnancy " of having excavated it.The later stage sixties, China was used for pregnant woman's induction of labour in second trimester and treatment hydatidiform mole clinically.U.S. scientist in 1989 has found that its can HIV inhibiting (HIV) duplicates and (has seen bibliographical information, Mcgrath MS, Hwang KM., Caldwell SE, et al in the T of acute infection lymphocyte and chronically infected scavenger cell.GLQ223：An?inhibitor?of?human?immunodefienfiency?virusreplication?in?acutely?and?chronically?infected?cells?of?lymphocyteand?mononuclear?phygocyte?lineage。And try out treatment clinically Proc Natl Acad SciUSA, 1989,86:2844～2848), in aids patient.We find also that with other scholars Trichosanthin also has to a certain degree anti-many other viruses, leukemia and resists the ability of other tumour (to see bibliographical information, Kong Mei, Ke Yibao, Zhou Meiyun etc.Trichosanthin brings out leukemia cell K562 Study of apoptosis.Journal of Experimental Biology, 1998,31 (3): 233～243.Zheng YT, Zhang KL, Ben KL, et al.In?vitro?immunotoxicity?and?cytotoxicityof?trichosanthin?against?human?nomal?immunocytes?andleukemia-lymphoma?cells。Immunopharmacology?andImmunotoxicology，1995，17(1)：69～79。Wu Yuxin, Xiang Danni, Zhang Shuiping etc.Trichosanthin is to the lethal effect and the Study on Mechanism thereof of gastrocolonic cancer cell.China's digestion magazine.1993。13(3)：263-266。)。But when Trichosanthin was used as drug inducing labor, the Chinese science man can cause the speed property sent out anaphylaxis by IgE mediation once in a while with regard to having found it, thereby each induced labor women can only inject once in life, had greatly limited its range of application.
The objective of the invention is to overcome above-mentioned weak point, develop the new Trichosanthin product that a kind of allergenicity is low, can use repeatedly.
The invention provides a kind of MUTANT OF TRICHO-SANTHIN product (MTCS), it is after transforming from the gene artificial mutation of natural trichosanthin, to utilize appropriate expression system to obtain.
Natural trichosanthin (TCS) is made up of 247 amino-acid residues, and the primary structure of its aminoacid sequence is as shown in the formula expression:
ATG?ATC?AGA
Met?Ile?ArgTTC?TTA?GTC?CTC?TCT?TTG?CTA?ATT?CTC?ACC?CTC?TTC?CTA?ACA?ACT?CCT?GCT?GTG?GAG?GGC↓Phe?Leu?Val?Leu?Ser?Leu?Leu?Ile?Leu?Thr?Leu?Phe?Leu?Thr?Thr?Pro?Ala?Val?Glu?Gly1GAT?GTT?AGC?TTC?CGT?TTA?TCA?GGT?GCA?ACA?AGC?AGT?TCC?TAT?GGA?GTT?TTC?ATT?TCA?AATAsp?Val?Ser?Phe?Arg?Leu?Ser?Gly?Ala?Thr?Ser?Ser?Ser?Tyr?Gly?Val?Phe?Ile?Ser?Asn
10??????????????????????????????????????20CTG?AGA?AAA?GCT?CTT?CCA?AAT?GAA?AGG?AAA?CTG?TAC?GAT?ATC?CCT?CTG?TTA?CGT?TCC?AGTLeu?Arg?Lys?Ala?Leu?Pro?Asn?Glu?Arg?Lys?Leu?Tyr?Asp?Ile?Pro?Leu?Leu?Arg?Ser?Ser
30??????????????????????????????????????40CTT?CCA?GGT?TCT?CAA?CGC?TAC?GCA?TTG?ATC?CAT?CTC?ACA?AAT?TAC?GCC?GAT?GAA?ACC?ATTLeu?Pro?Gly?Ser?Gln?Arg?Tyr?Ala?Leu?Ile?His?Leu?Thr?Asn?Tyr?Ala?Asp?Glu?Thr?Ile
50?????????????????????????????????????60TCA?GTG?GCC?ATA?GAC?GTA?ACG?AAC?GTC?TAT?ATT?ATG?GGA?TAT?CGC?GCT?GGC?GAT?ACA?TCCSer?Val?Ala?Ile?Asp?Val?Thr?Asn?Val?Tyr?Ile?Met?Gly?Tyr?Arg?Ala?Gly?Asp?Thr?Ser
70?????????????????????????????????????80TAT?TTT?TTC?AAC?GAG?GCT?TCT?GCA?ACA?GAA?GCT?GCA?AAA?TAT?GTA?TTC?AAA?GAC?GCT?ATGTyr?Phe?Phe?Asn?Glu?Ala?Ser?Ala?Thr?Glu?Ala?Ala?Lys?Tyr?Val?Phe?Lys?Asp?Ala?Met
90?????????????????????????????????????100CGA?AAA?GTT?ACG?CTT?CCA?TAT?TCT?GGC?AAT?TAC?GAA?AGG?CTT?CAA?ACT?GCT?GCA?GGC?AAAArg?Lys?Val?Thr?Leu?Pro?Tyr?Ser?Gly?Asn?Tyr?Glu?Arg?Leu?Gln?Thr?Ala?Ala?Gly?Lys
110?????????????????????????????????????120ATA?AGG?GAA?AAT?ATT?CCG?CTT?GGA?CTC?CCT?GCT?TTG?GAC?AGT?GCC?ATT?ACC?ACT?TTG?TTTIle?Arg?Glu?Asn?Ile?Pro?Leu?Gly?Leu?Pro?Ala?Leu?Asp?Ser?Ala?Ile?Thr?Thr?Leu?Phe
130?????????????????????????????????????140TAC?TAC?AAC?GCC?AAT?TCT?GCT?GCG?TCG?GCA?CTT?ATG?GTA?CTC?ATT?CAG?TCG?ACG?TCT?GAGTyr?Tyr?Asn?Ala?Asn?Ser?Ala?Ala?Ser?Ala?Leu?Met?Val?Leu?Ile?Gln?Ser?Thr?Ser?Glu
150?????????????????????????????????????160GCT?GCG?AGG?TAT?AAA?TTT?ATT?GAG?CAA?CAA?ATT?GGG?AAG?CGT?GTT?GAC?AAA?ACC?TTC?CTAAla?Ala?Arg?Tyr?Lys?Phe?Ile?Glu?Gln?Gln?Ile?Gly?Lys?Arg?Val?Asp?Lys?Thr?Phe?Leu
170?????????????????????????????????????180CCA?AGT?TTA?GCA?ATT?ATA?AGT?TTG?GAA?AAT?AGT?TGG?TCT?GCT?CTC?TCC?AAG?CAA?ATT?CAGPro?Ser?Leu?Ala?Ile?Ile?Ser?Leu?Glu?Asn?Ser?Trp?Ser?Ala?Leu?Ser?Lys?Gln?Ile?Gln
190?????????????????????????????????????200ATA?GCG?AGT?ACT?AAT?AAT?GCA?CAG?TTT?GAA?AGT?CCT?GTT?GTG?CTT?ATA?AAT?GCT?CAA?AACIle?Ala?Ser?Thr?Asn?Asn?Gly?Gln?Phe?Glu?Ser?Pro?Val?Val?Leu?Ile?Asn?Ala?Gln?Asn
210?????????????????????????????????????220CAA?CGA?GTC?ACG?ATA?ACC?AAT?GTT?GAT?GCT?GGA?GTT?GTA?ACC?TCC?AAC?ATC?GCG?TTG?CTGGln?Arg?Val?Thr?Ile?Thr?Asn?Val?Asp?Ala?Gly?Val?Val?Thr?Ser?Asn?Ile?Ala?Leu?Leu
230?????????????????????????????????????240CTG?AAT?AGA?AAC?AAT?ATG?GCA↓GCC?ATG?GAT?GAC?GAT?GTT?CCT?ATG?ACA?CAG?AGC?TTTLeu?Asn??Arg?Asn?Asn?Met?Ala?Ala?Met?Asp?Asp?Asp?Val?Pro?Met?Thr?Gln?Ser?Phe
247GGA?TGT?GGA?AGT?TAT?GCT?ATT?TAGGly?Cys?Gly?Ser?Tyr?Ala?Ile?End
Numeral amino acid sequence number in the following formula
The primary structure of MTCS is formed the most aminoacid sequences that kept natural TCS structure.One of its feature is this proteinic the 174th change to 180 7 amino-acid residues, one of change to any polare Aminosaeren residue more than 1 or 1 in 4 polare Aminosaeren residues is wherein lacked or be transformed into the nonpolar amino acid residue, and the change of acid and alkaline amino acid residue; Two of change is that 3 nonpolar amino acid residues in these 7 amino-acid residues are lacked arbitrarily; Two of constitutional features is that the carboxyl terminal of TCS done the disappearance or the transformation of any two or more polare Aminosaeren residues in 14 polare Aminosaeren residues in the individual amino-acid residue of 22-45 (being that sequence number is 226-203) in due order, the change and the polarity that comprise acidity, alkaline amino acid residue arrive nonpolar transformation.Become new MUTANT OF TRICHO-SANTHIN discrepant with natural TCS structure, that have excellent in performance, it has greatly reduced the allergenicity of Trichosanthin, but has kept all biologic activity of TCS.
The change of this product primary structure, the notion of the polare Aminosaeren that relates to are meant Serine (Ser), Threonine (Thr), halfcystine (Cys), tyrosine (Tyr), aspartic acid (Asp), asparagine (Asn), L-glutamic acid (Glu), glutamine (Gln), Methionin (Lys), arginine (Arg), Histidine 11 seed amino acids such as (His).Wherein, Asp, Asn, Glu, Gln are acidic amino acid, and Lys, Arg, His are basic aminoacids; The notion of nonpolar amino acid is meant glycine (Gly), L-Ala (Ala), Xie Ansuan (Val), leucine (Leu), Isoleucine (Ile), proline(Pro) (Pro), phenylalanine (Phe), tryptophane (Try), methionine(Met) nine seed amino acids such as (Met).
The immunologic competence and the efficacy testing result of MUTANT OF TRICHO-SANTHIN product of the present invention (MTCS) are as follows:
One, the character of TCS excalation albumen and mutain
In order to explore the relation of primary structure and biologic activity and immunologic competence, from gene level TCS is transformed, obtain a plurality of amino-acid residues and transformed the gene that lacks with C-terminal, behind escherichia coli expression, purifying, obtained the protein that corresponding a plurality of amino-acid residue is transformed and C-terminal lacks respectively.Because TCS and mutant thereof are a kind of RNAN-candy glycosides enzymes, can make the rrna inactivation, thereby blocking protein is synthetic, thereby after these mutant and C-terminal have the serial proteic biology and immunologic competence of the mutant that clearly lacks the position after testing, in conjunction with the space Structure Analysis of TCS, make the architecture basics of active region and judge data.The RIP activity is according to Pelhem ﹠amp; The method of Jackson is carried out; The antifertility activity is undertaken by our breadboard ordinary method, and external immune response detects with the Elisa method.
Shown in experimental result can be listed as follows:
Product The position is changed in C end AA change disappearance position The RIP activity * The antifertility activity ** External immune response ability IgG *** External immune response ability IgE ***
NTCS 1-247 ???0??????????????0 ????++ ????++ ????++ ????++
L 3TCS 1-244 247-244???????????0 ????++ ????++ ????++ ????++
L 10TCS 1-237 247-239???????????0 ????++ ????++ ????++ ????++
L 14TCS 1-232 247-233???????????0 ????++ ????++ ????+ ????+
L 29TCS 1-218 247-219???????????0 ????+ ????+ ????± ????±
L 46TCS 1-201 247-202???????????0 ????+ ????+ ????± ????±
L 52TCS 1-195 247-196???????????0 ????± ????± ?????- ?????-
L 67TCS 1-180 247-181???????????0 ?????- ????- ?????- ?????-
L 74TCS 1-173 247-174???????????0 ?????- ????- ?????- ?????-
L 11TCS 1-173，181-247 174-180???????????0 ????± ????± ????± ????±
L 52TCS 1-173，181-202 247-203，180-174??0 ?????- ????- ?????- ?????-
M 7TCS 1-247 ??0??????????? 180-174 ????++ ????++ ????± ????±
M 24TCS 1-247 ??0??????????? 226-203 ????++ ????++ ????± ????±
M 31TCS 1-247 ??0???? 226-203，180-174 ????++ ????++ ????- ?????-
Annotate: NTCS is natural TCS; LTCS is deletant TCS; MTCS is mutant TCS. *The active IC of RIP 50(ng/ml) :≤50++,＜50 → 〉=500+,＜500 → 〉=5000 ±,＞5001-; *Active the %: 〉=80++ of antifertility,＜80 → 〉=30+,＜30 → 〉=5 ±,＜5-; * *External immune response ability %: 〉=80++,＜80 → 〉=30+,＜30 → 〉=10 ±,＜10-.
At M 31TCS (M226-203,180-174)In this mutant, two sudden change zones are arranged, sequence 174-180 and sequence 203-226, wherein the polare Aminosaeren residue that can be suddenlyd change or lack is totally 18, totally 3 of nonpolar amino acid residues.These two zones one or more (174-180) can be arranged respectively or more than two or two the polare Aminosaeren residue of (203-226) suddenlyd change and transformed or disappearance, also can have 3 nonpolar amino acid residues to be lacked in the 174-180 zone.
The amino acid whose sequence distribution list that the 174-180 zone can be suddenlyd change is:
Original acid residue and sequence number thereof The amino-acid residue that preferably is transformed into
Arg 174、 Asp 176Lys 177Thr 178Val 175Phe 179leu 180 Glu, Asp, Gly Lys, Gly Glu, Asp, Gly Gly, Ala disappearance disappearance disappearance
The amino acid whose sequence distribution list that the 203-226 zone can be suddenlyd change is:
Original acid residue and sequence number thereof The amino-acid residue that preferably is transformed into
Ser 203、Ser 211Thr 204、Thr 224、Thr 226Asn 205、Asn 206、Asn 217、Asn 220Gln 208、Gln 219、Gln 221Glu 210Arg 222 ????Gly、Ala ????Gly、Ala ????Lys、Gly ????Lys、Gly ????Lys、Gly ????Lys、Gly
The zone Preferred two mutational site
??174-180 ??203-226 ????Arg 174With Asp 176Or Lys 177With Thr 178????Ser 203With Thr 204Or Glu 210With Thr 226
The main character detected result of the mutein (M-TCS) that deletant protein (L-TCS) that obtains from the said gene disappearance and transgenation obtain shows: along with being enlarged to gradually of C-terminal amino-acid residue disappearance degree lacks 10 amino-acid residues, their IgG also presents strong positive as NTCS with IgE vitro detection immunocompetence; After the disappearance degree was enlarged to 14 amino-acid residues, immunocompetence then descended to some extent, but rrna inactivation and two kinds of biologic activity that cause pregnant mouse induction of labour in second trimester are not affected as yet.Until 29 amino-acid residues of disappearance, its biologic activity just is affected.Described in the past by the author, the biologic activity position of TCS is in sequence 110 to 174 zones, but the lax variation of the space structure that end amino acid disappearance to a certain degree causes is still influential to the active region, and this amino-acid residue disappearance is more near the zone, active centre, inactivation is big more, until losing whole activity.After the immunocompetence zone is arranged in than the biologic activity domain structure, compare, thereby early be subjected to the structure influence of aminoacid deletion near C-terminal.After lacking 29 amino-acid residues, its immunocompetence has been reduced to the faint positive.The external immune response relevant with IgG and IgE is reduced to faint male product and still keeping and comprise that RIP and two kinds of active protein of antifertility have L 29TCS 1-218, L 46TCS 1-201, and still keep two kinds of active M of having of strong biology 7TCS (M180-174)And M 24TCS (M226-203)Two kinds, two kinds of external immunocompetences are negative and keep still that two kinds of strong biology is active to be M 31TCS (M226-203,180-174)A kind of.This is that a kind of character is particularly outstanding, has greatly reduced the immunogenicity of natural trichosanthin (NTCS), also almost can't harm the new MUTANT OF TRICHO-SANTHIN of biologic activity.Two, the character of M-TCS:
The outer inactivation rrna ability of detection bodies, conceived small white mouse induction of labour in second trimester ability, external and IgG and IgE response capacity and under vitro culture the situation of killing to different cells be our breadboard conventional application method.
1. compare with biologic activity with the immunity of natural TCS through improved MTCS product with preferred properties.Summary sees the following form:
NTCS ????MTCS
Inactivation ribosomes ability (%) induction of labour in second trimester ability (%) cytotoxicity (K562 cell line) is external to be brought out in the big white mouse body IgE situation of replying with IgG respond (%) and brings out the acute irritated situation of cavy with IgE respond (%) is external ??100 ??100 ??100 ??100 ??100 ???++ ???++ 100 100 100＜10＜10-(seeing 3d) ± (seeing 3c)
2.MTCS the situation of killing to different cells is summarized in following table:
????CELL?LINE ?LC 50(ug/ml) CELL?LINE ??LC 50(ug/ml) CELL?LINE ?LC 50(ug/ml)
????Jar ????HL60 ????MLT ????K562 ????U937 7.0 8.1 10.0 11.0 17.0 ????B16 ????A431 ????B7-325 ????SPCA1 ????7404 ????69.2 ????77.6 ????218.8 ????257.0 ????354.8 ????Wish ?? *HMPLC ??＞1000 ??＞1000
*Behaviour normal circumference blood lymphocyte is from clinical collection.
As seen, MTCS is to half sterilizing dose LC of leukemia cells such as K562, HL60, MLT, U937 and Jar people's suede cancer cells from last table 50＜30ug/ml, we claim LC 50The cell of≤30ug/ml is the most responsive cell mass; MTCS is to the LC of B16, A431, B7-325, SPCA1, cancer cells such as 7404 50More than＞the 30ug/ml, claim that they are general responsive type cell mass; MTCS is to the LC of normal somatic cells such as human peripheral lymphocyte and human amniotic cell 50More than＞the 1000ug/ml, be called non-sensitive type cell mass.This is just clearly clear and definite under certain dosage condition, and MTCS has the intensive killing action to cancer cells such as leukemia; And it is insensitive to normal somatic cell.This is consistent with the research to TCS in the past.
3, the character of anti-human body animal model for cancer.
a)。Pharmacodynamic study.
With human leukemia red white corpuscle cancer make nude mouse (nodemice) subcutaneous in K562 cell inoculation to eight week, inoculating cell quantity is 1 * 10 7The order of magnitude.Inoculate after 10 days, visible tumour generates at inoculation position, and success ratio of inoculation is 50%.Select tumour to generate mouse and be divided into four groups at random, give the MTCS injection for curing of various dose.And in contrast with physiological saline injection mouse.Under every mouse therapeutic dose≤induced labor dosage (15nm/Kg, 45nm/Kg, 75nm/Kg) condition, injection in per four days is once exceeded with four treatments, all visible in various degree but the knurl effect.The tumour inhibiting rate of each treatment group is respectively (40 ± 3) %, (71 ± 2) % and (92 ± 4) %.Select the two deficient animals Scid mouse human body animal models of another kind of immunity for use, continue the killing effect of research MTCS red white corpuscle cancer (K562).Select for use to make the Scid mouse give K562 cell inoculation in nine weeks, the same nude mouse of inoculating cell quantity, success ratio of inoculation is 100%.After inoculation the 5th day, mouse is divided into four groups at random, wherein three groups is MTCS treatment group, another group is physiology saline control group.The treatment group is carried out injection for curing by high, medium and low various dose.Be divided into 150nm/kg shot group, 75nm/kg secondary treatment injection group (injection once weekly) and 37nm/kg injection group (injection in per four days once), total dose is 150nm/kg.The experiment conclusion that high, medium and low three groups of tumour inhibiting rates are respectively (43 ± 6) %, (64 ± 8) % and (94 ± 3) %, two kinds of human body animal models is under induced labor dosage condition, the just visible knurl effect that significantly presses down; Four just visible tumor killing effects more than 90% of injection of half induced labor dosage, promptly low dose of multiple injection has better effect.
Above-mentioned for after the cancer cells of people's red white corpuscle leukemia cell cancer is seeded to two kinds of animal bodies and forms solid tumors, the result of treatment of MTCS is all good.On the basis of these two kinds of animal models, continue to have set up the animal model of the more approaching human leukemia of a kind of and clinical manifestation with the Scid mouse, promptly a kind ofly can reflect the leukemic mouse in people source from the variation of small white mouse blood parameters, white blood cell count.Experiment is from the MTCS of mouse tail vein injection 3.75nm/kg, 7.5nm/kg and 15nm/kg dosage, the mouse that suffers from people's red white corpuscle cancer in order to treatment, after three injections, the mouse that above-mentioned three groups of white cell sums return to normal value is respectively (25 ± 6) %, (75 ± 8) % and (94 ± 3) %.The dosage that is MTCS treatment liquid tumor can be less than solid tumor, and the effect of treatment liquid tumor is better than solid tumor.
b)。The research of small white mouse acute toxicity.
Select eight week ICR/JCL-F1 mouse in age studies on acute toxicity method routinely for use, shot MTCS and NTCS, the relatively LD of MTCS and NTCS 50, the result shows:
Under same dosage condition, natural group of animal dead rate is greater than the mutant group, and natural group of death time of animal is early than the mutant group.With seven days be observing time, their medial lethal dose is respectively LD 50(NTCS)=18.4mg/kg and LD 50(MTCS)=27.5mg/kg, thereby the acute toxicity of MTCS is starkly lower than NTCS, descended 50% approximately.
c)。Acute irritated toxicity test in the cavy body.
NTCS and MTCS are made acute irritated toxicity test in the cavy body respectively.The sensitization consumption is 4.5 multiple doses that an intradermal injection causes pregnant woman's clinical dosage, attacks after 14 days, and challenge dose is that the injection of ear vein causes miscarry 10 times of dosage of pregnant woman.Reaction and the death condition of animal in 30 minutes looked in sight.NTCS group dead animal number is 12/14 with the ratio of experimental animal sum as a result; Account for 86%, the MTCS group is 3/15, accounts for 20%, and both compare significant difference.Be in the cavy body that causes of MTCS acute irritated degree than the remarkable reduction of NTCS.
d)。The models of passive skin irritability of rats test.
Press Ovary, the method for Z.S. is done models of passive skin irritability of rats (Passive Cutaneousanaphylaxis, PCA) test.With N-TCS and M-TCS difference sensitization small white mouse, after 14 days, get the antiserum(antisera) of NTCS and MTCS respectively.Again two kinds of antiserum(antisera)s are injected the big white mouse of having anaesthetized respectively and shave plucked back intracutaneous, and the tail vein of using the Evans Blue that contains NTCS and MTCS to be injected into big white mouse is respectively attacked, after half an hour, draw tail to cause death, sight is looked into big white mouse skin of back locus coeruleus and formed situation, and is positive greater than 0.5cm person with the locus coeruleus diameter.The result shows that the rat back patch through the injection of N-TCS antiserum(antisera) all shows positive (+), and the rat back patch of M-TCS antiserum(antisera) injection all shows negative (-).Show that MTCS brings out in the big white mouse body IgE and replys and fall sharply, the positive reaction of former NTCS all disappears.Three, the research of the antitumor mechanism of MTCS:
1.MTCS induced the apoptosis of responsive tumour cell.
As described in top second the 1st and the 2nd, MTCS has extremely strong killing action to the leukemia cell in the vitro culture, and atomic to normal impact cell.We are example with MTCS to the killing action of K562 cell, through multiple research means: typical apoptosis morphological change has taken place in the K562 cell after the visible dosing by electron microscope observation, cell diminishes, endochylema concentrates, reticulum dilatation, and kernel disappears, one of chromatin condensation simmer down to or several agglomerates, sometimes can form crescent, cavity occur in many cells, be formed with adventitia at last and hold typical apoptotic body.Extracting dosing group cell DNA on the biochemistry, with the appearance of agarose coagulation electrophoresis observation to " stepped " band, this is the biochemical indicator of apoptosis.And the diploid peak (being apoptotic peak) that utilizes the detected apoptotic cell of flow cytometer (FACS) to occur, all results show that all TCS has induced the apoptosis of K562 cell.
2. exist on the cytolemma of sensitive cells can with the single-minded bonded protein ingredient of MTCS.
(Biomolecular InteractionAnalysis, BIA) research when the cytolemma extract of the sensitive cells bio-sensing sheet of flowing through, can combine with immobilised MTCS, causes the resonance on the sensing chip to change with the biomolecular interaction analysis system.This variation is converted to spr signal and weighs with RU (Resonance unite).Sensitive cells all has 100 to 300 RU that do not wait to change, and it is very strong to show that MTCS combines with the macromole of membranin.And handle normal cell such as people uterus amnion cell equally, then there is not this variation.
3. by laser confocal scanning microscope (LCSM) research, observe MTCS and can be arrived in the K562 cell cytosol by endocytosis by receptor-mediated form;
4. pass through [ 35S] GTP γ TCS is in conjunction with experiment, illustrates that MTCS can activate the G albumen on the sensitive cells film, and insensitive cell does not then have this and activates phenomenon.Show the generation and the transduction that in sensitive cells, have caused signal;
5. by laser confocal scanning microscope (LCSM) research, after further observing external source MTCS and stimulating sensitive cells, can cause the release and the change in concentration of free calcium ion in the intracellular calcium store.
The conclusion that draws according to above research is: exist the acceptor of MTCS on the cytolemma of sensitive cells, can mediate MTCS and enter in the cell, performance causes rrna inactivation (RIP) effect; Simultaneously, MTCS has also disturbed the signal transduction of cell.Above-mentioned dual function has caused the apoptosis of cell.These sensitive cells thumping majorities are tumour cells, thereby MTCS treatment cancer is with antitumor drug is different fully with the internecine kill mechanism of normal cell to tumour cell usually.For the MTCS conduct newly will provide solid theory for the clinical application of antitumor drug.
Another object of the present invention has provided the preparation method of above-mentioned MUTANT OF TRICHO-SANTHIN product, and this method comprises the following steps: (1) transformation to former TCS gene
The gene of natural trichosanthin is that (termination codon) amounts to 870bp (base pair) from ATG (initiator codon) to TAG, can get from gene pool or cDNA storehouse sieve, or utilize PCR method to hang and get, also can utilize chemical process manually complete synthesis, this genes encoding a precursor protein of forming by 289 amino-acid residues, be it except the TCS (247 amino-acid residue) of encoding mature, before its 5 ' end, also encoded a leading peptide of forming by 23 peptides or signal peptide and at its 3 ' also encoded after holding an extension peptide or tail peptide (19 peptide);
Utilize all rite-directed mutagenesis methods of known DNA of biology field worker, comprise that single-stranded template method and double-stranded template method (being the PCR method) are to the natural TCS gene of primary transformation: a. introduction restriction enzyme Nco I site (CCATGG) before 5 ' end of the dna sequence dna of encoding mature TCS that suddenlys change, also just introduce initiator codon ATG, added a methionine(Met) before the N of the former mature T CS that will encode the simultaneously end thus; Or introduce restriction enzyme NdeI site (CATATG), make first amino-acid residue of the former mature T CS of coding change methionine(Met) into thus simultaneously from aspartic acid;
Restriction enzyme BamHI site (GGATCC) is introduced in 3 ' end back at the dna sequence dna of encoding mature TCS, has introduced terminator codon thus simultaneously; B. to appropriate position (174-180,203-226) amino-acid residue is transformed, to obtain the gene of MUTANT OF TRICHO-SANTHIN of the present invention; (3) expression of MTCS
The gene that will have the MUTANT OF TRICHO-SANTHIN of excellent in performance advances plasmid vector pET-2d (or pET-3a) with Nco I (or Nde I) and Bam HI double digestion rear clone, and ordinary method transformation receptor bacterium intestinal bacteria Bl 21 (DE3, plysS).Transformant (contains Ap at 37 ℃ with the LB nutrient solution, Chl) in after the overnight incubation, (containing Ap, is about 0.8 in same temperature amplification fermentation culture to the photoabsorption of 570mu Chl) with the M9ZB nutrient solution, adding inductor IPTG is 0.5mM to final concentration, and centrifugal collection thalline after 3 hours is cultivated in continuation.Thalline after ultrasonication, CM-Sephadex or CM-Sephorose column chromatographic isolation and purification on the centrifuging and taking supernatant liquor, its main peak is the MTCS product.
Product of the present invention can be used for clinical medicine.
Except that all indications that comprised former natural trichosanthin, also be extended to antitumor field, promptly can be applicable to:
1) anti-other noumenal tumour of leukemia and broad spectrum.Its advantage is optionally to enter target cell, thereby consumption is little, and access times are few.In certain dosage range, tumour cell there is strong lethal effect, and injuring normal cell hardly, so untoward reaction is atomic.
2) antiviral, anti-HIV can be used for the treatment of aids patient, and more natural TCS side reaction is few, and patient can repeatedly use.
3) gravid woman's induction of labour in second trimester, ectopic pregnancy can repeatedly be used.
Embodiment 1
The rite-directed mutagenesis of TCS gene and clone.
For making things convenient for genetic manipulation, the primer of design has:
1.5’GTGCAGGCC?ATG?GAT?GTTAGG?3’
2.5’AAC?AAT?ATG?GCA?TAG?GAT?CCC?ATG?GAT?GAC?3’
The characteristics of primer 1 are to contain Nco I site (CCATGG), introduce initiator codon (ATG) simultaneously.The result has added a Met before the N of the former mature T CS of coding end;
The characteristics of primer 2 are to contain BamH I site (GGATCC), and the result introduces terminator codon (TAG) behind the TCS of encoding mature.
The Muta-Gene Phagemid in vitromutagenesis medicine box of selecting for use Bio-Rad company to provide carries out mutation operation by detailed description appended in the medicine box and step, meets test design by the dna sequence analysis checking gene that obtains at last.This method require earlier will be to be suddenlyd change the original gene precursor clone into phagemid (phagemid) series.We have selected pTZ-19U.
With above-mentioned with the good pTZ-19U-TCS of primer 1 sudden change through Nco I and Bam HI 37 ℃ of hydrolysis 2 hours, by low melting point agarose coagulation electrophoretic separation and reclaim the TCS gene fragment of the about 750bp of length in the TAE damping fluid, utilize T 4Dna ligase spends the night with prior same the connection through the expressive plasmid pET-2d of Nco I and BamH I double enzymolysis at 4 ℃, obtains pET-2d-TCS.
Embodiment 2
PET-2d-TCS transformation receptor bacterium and expression thereof
The conventional method transformed into escherichia coli BL21 of pET-2d-TCS (DE3, plysS).Transformant (contains Ap at 37 ℃ with the LB nutrient solution, Chl) in after the overnight incubation, (contain Ap, Chl) amplifying fermentation culture to the photoabsorption of 600mu in same temperature is about 0.8 to use the M9ZB nutrient solution again, adding inductor IPTG is 0.5mM to final concentration, and centrifugal collection thalline after 3 hours is cultivated in continuation.Thalline after ultrasonication, CM-Sephadex or CM-Sephorose column chromatography on the centrifuging and taking supernatant liquor.Utilization contains the Nacl separation and purification of (0.1-0.4) M concentration gradient of 50mM Tris-Hcl damping fluid, and its main peak is the product of expression.
Embodiment 3
The gene clone of MTCS.
Remove according to needs, outside the primer difference of design, all the other operations are all identical with top embodiment 1.Obtain containing the plasmid pET2d-MTCS of MTCS gene at last.
With MTCS (M177,203,204)Be example, the mutant primer of design is:
3.?5’AAG?CGT?GTT?GAC? GAA?ACC?TTC?CTA?CCA?3’
4.?5’ATT?CAG?ATA?GCG? GGA?GGT?AAT?AAT?GGA?CAG?3’
The characteristics of primer 3 are the codons that the codon of former 177 Lys changed over Glu.Because the merger of codon, the base at underscore position can also substitute with the bases G AG of other Glu that encodes.
The characteristics of primer 4 are the codons that the codon of former 203 and 204 Ser and Thr changed over Gly and Gly.Because the merger of codon, the base at underscore position can also with the base of other Gly that encodes as: GGG GGG, GGA GGA, GGC GGC, GGT GGT etc. have 16 kinds of alternative.
Embodiment 4
The conversion of MTCS gene and expression.
The plasmid pET2d-MTCS method transformed into escherichia coli BL21 of routine that contains the MTCS gene (DE3, plysS).The used method of the conversion of MTCS gene, expression and purifying is identical with the foregoing description 2.Obtain required mutant TCS at last, in the case of this example, be MTCS (M177,203,204).
Embodiment 5
MTCS (M177,203,204)Biologic activity and immunocompetent detection.
RIP is active to be undertaken by document [8] report method, and rabbit reticulocyte lysate is the Promega product, [ 3H] leucine is New England Nuclear product.Antifertility is active to carry out [9] with our laboratory ordinary method, is model with 11 days ICR mouse of pregnancy in mid-term, back injection MTCS (M177,203,204)The 75nM/kg drug dose, disconnected neck is put to death mouse after 48 hours.Dissect total tire mouse number of record and dead tire mouse number (comprising the fetus absorption point), calculate peptide mouse mortality ratio.External immunocompetence detects with the Elisa method, and IgG antibody and monoclonal antibody IgE prepare and purifying [10] voluntarily for our laboratory.The purification process affinity column, wherein TCS-Sepharose 4B is by the preparation of cyanogen bromide-activated method.The gel electrophoresis of SDS-polypropylene amine is undertaken by the Laemmli method, examines the bright blue R250 dyeing of Ma Shi with 0.25% behind the electrophoresis.More than every experimental result except that negative control, all use the positive contrast of NTCS.The experimental result brief summary is:
Product Amino acid is changed point RIP activity (%) Antifertility activity (%) External immunity External immunity
Response capacity IgG Response capacity IgE
NTCS MTCS ???????0 ?177，203，204 ????100 ????100 ????100 ????100 ????100 ????＜10 ????100 ???＜10
Embodiment 6
MTCS (M177,203,204)Killing effect to the K562 cell
MTT method with the laboratory routine is measured MTCS (M177,203,204)Killing effect to the K562 cell.People's red white corpuscle type leukemia K 562 with pick up from clinical isolating normal people's lymphocyte, respectively in the RPMI-1640 nutrient solution that contains 10% deactivation calf serum, under 37 ℃, 5%CO2 condition, with 2 * 10 5/ ml inoculating cell, and add 1,5,10,20,50 respectively, the MTCS of 100ug/ml.Cultivate that every hole adds 25ulMTT (available from Sigma) (5mg/ml) after 48 hours, every hole adds Lysing Buffer termination reaction after 2 hours, and 370 ℃ of placements are spent the night, and measure each hole A with microplate reader 570Value.End-result shows the LC of MTCS to K562 50Be 11.0ug/ml, and insensitive to human peripheral lymphocytes, the concentration of above-mentioned test dose MTCS up to the 100ug/ml condition under, can not survey LC 50Value.

Claims (3)

1, a kind of MUTANT OF TRICHO-SANTHIN product, it is characterized in that this product has changed the 174th to 180 7 amino-acid residues of Trichosanthin matter, and wherein any one in 4 polare Aminosaeren residues or more than one polare Aminosaeren residue are lacked or are transformed into the nonpolar amino acid residue, and the change of acid and alkaline amino acid residue; Two of change is that 3 nonpolar amino acid residues in these 7 amino-acid residues are lacked arbitrarily; And the disappearance or the transformation of any two or more polare Aminosaeren residues in 14 polare Aminosaeren residues in the individual amino-acid residue of 22-45 (being that sequence number is 226-203) have been done in due order at the carboxyl terminal of TCS, comprise the change of acidity, alkaline amino acid residue, arrive nonpolar transformation with polarity, become new MUTANT OF TRICHO-SANTHIN discrepant with natural TCS structure, that have excellent in performance.
2, a kind of MUTANT OF TRICHO-SANTHIN according to claim 1, it is characterized in that the change of wherein said primary structure, the polare Aminosaeren that relates to is meant Serine (Ser), Threonine (Thr), halfcystine (Cys), tyrosine (Tyr), aspartic acid (Asp), asparagine (Asn), L-glutamic acid (Glu), glutamine (Gln), Methionin (Lys), arginine (Arg) or Histidine (His).Wherein, Asp, Asn, Glu, Gln are acidic amino acid, and Lys, Arg, His are basic aminoacids; Nonpolar amino acid is meant glycine (Gly), L-Ala (Ala), Xie Ansuan (Val), leucine (Leu), Isoleucine (Ile), proline(Pro) (Pro), phenylalanine (Phe), tryptophane (Try) or methionine(Met) (Met).
3, a kind of preparation method of MUTANT OF TRICHO-SANTHIN product as claimed in claim 1 is characterized in that this method comprises the following steps: (1) transformation to former TCS gene
Utilize all rite-directed mutagenesis methods of known DNA of biology field worker, comprise that single-stranded template method and double-stranded template method (being the PCR method) are to the natural TCS gene of primary transformation: a. introduction restriction enzyme Nco I site (CCATGG) before 5 ' end of the dna sequence dna of encoding mature TCS that suddenlys change, introduce initiator codon thus, added a methionine(Met) before the N of the former mature T CS that will the encode end simultaneously; Or introduce restriction enzyme Nde I site (CATATG), make first amino-acid residue of the former mature T CS of coding change methionine(Met) into thus simultaneously from aspartic acid;
Restriction enzyme BamHI site (GGATCC) is introduced in 3 ' end back at the dna sequence dna of encoding mature TCS, has introduced terminator codon thus simultaneously; B. to appropriate position (174-180,203-226) amino-acid residue is transformed, to obtain the gene of MUTANT OF TRICHO-SANTHIN of the present invention; (2) expression of MTCS
The gene that will have the MUTANT OF TRICHO-SANTHIN of excellent in performance advances plasmid vector pET-2d (or pET-3a) with Nco I (or Nde I) and Bam HI double digestion rear clone, ordinary method transformation receptor bacterium intestinal bacteria Bl 21 (DE3, plysS), transformant (contains Ap at 37 ℃ with the LB nutrient solution, Chl) after the middle overnight incubation, (contain Ap with the M9ZB nutrient solution, Chl) amplifying fermentation culture to the photoabsorption of 570mu in same temperature is about 0.8, add inductor IPTG to final concentration be 0.5mM, continue to cultivate centrifugal collection thalline after 3 hours, thalline is after ultrasonication, CM-Sephadex or CM-Sephorose column chromatographic isolation and purification on the centrifuging and taking supernatant liquor, its main peak is the MTCS product.
CN 00119553 2000-08-02 2000-08-02 Trichosanthin mutant and its preparing process Pending CN1283630A (en)

Priority Applications (9)

Application Number Priority Date Filing Date Title
CN 00119553 CN1283630A (en) 2000-08-02 2000-08-02 Trichosanthin mutant and its preparing process
CNB011031026A CN1152051C (en) 2000-08-02 2001-01-18 Trichosanthin mutant and its prepn.
US09/905,247 US7157552B2 (en) 2000-08-02 2001-07-13 Mutant trichosanthin
EP01983407.6A EP1307480B1 (en) 2000-08-02 2001-07-18 Mutant trichosanthin
PCT/CN2001/001178 WO2002012537A2 (en) 2000-08-02 2001-07-18 Mutant trichosanthin
CNB018167012A CN1208343C (en) 2000-08-02 2001-07-18 Trichosanthin mutant
AU2002214919A AU2002214919A1 (en) 2000-08-02 2001-07-18 Mutant trichosanthin
JP2002517821A JP4418155B2 (en) 2000-08-02 2001-07-18 Mutant heaven pollen protein
HK04104880A HK1061872A1 (en) 2000-08-02 2004-07-06 Mutant trichosanthin.

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1312175C (en) * 2004-09-09 2007-04-25 北京大学 Trichosanthin mutant and coding gene thereof
CN101469016B (en) * 2007-12-29 2011-09-07 上海交通大学医学院 Trichosanthin protein derived peptide and use thereof
CN103724409A (en) * 2012-10-15 2014-04-16 上海交通大学医学院 Trichosanthin effective epitope peptide fragment with immunosuppression effect and application thereof
CN105886579A (en) * 2014-11-27 2016-08-24 无锡翔天奇生物科技有限公司 Fermentation process and purifying preparation method of mutant of gene recombinant trichosanthin (MTCS)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1312175C (en) * 2004-09-09 2007-04-25 北京大学 Trichosanthin mutant and coding gene thereof
CN101469016B (en) * 2007-12-29 2011-09-07 上海交通大学医学院 Trichosanthin protein derived peptide and use thereof
CN103724409A (en) * 2012-10-15 2014-04-16 上海交通大学医学院 Trichosanthin effective epitope peptide fragment with immunosuppression effect and application thereof
CN103724409B (en) * 2012-10-15 2015-06-10 上海交通大学医学院 Trichosanthin effective epitope peptide fragment with immunosuppression effect and application thereof
CN105886579A (en) * 2014-11-27 2016-08-24 无锡翔天奇生物科技有限公司 Fermentation process and purifying preparation method of mutant of gene recombinant trichosanthin (MTCS)

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