CN105255929A - Method for increasing expression quantity of human metallothionein (MT4) prokaryotic expression vector - Google Patents

Abstract

The invention discloses a method for increasing expression quantity of a human metallothionein (MT4) prokaryotic expression vector. The method includes the steps that a DNA molecule of MT4 optimized by a codon is artificially synthesized, and the molecule further comprises DNA incision enzyme recognition sequences NcoI, SpeI and NheI; the DNA molecule is built in an NcoI/NheI cloning region of pET28a(+); by means of a series of reconstruction, the encoding gene of MT4 containing a ribosome binding region is expanded to 4 copies. The method is suitable for building expression vectors of all micro-molecule polypeptides (within 100 amino acids), the whole process is easy and quick to operate, the expression quantity of MT4 protein is increased through the prepared prokaryotic expression vector, cost is reduced, and work efficiency is improved.

Description

A kind of method improving human metal thioalbumen MT4 prokaryotic expression carrier expression amount

Technical field

The invention belongs to genetically engineered field, particularly relate to a kind of method improving human metal thioalbumen MT4 prokaryotic expression carrier expression amount.

Background technology

Metallothionein(MT) (Metallothionein, MT), also known as sulfydryl metal binding protein., except the MT containing cadmium (Cd), zinc (Zn), also there is the MT containing copper (Cu), mercury (Hz), element such as gold (Au), bismuth (Bi) etc. at nature in one class non-enzymatic protein.Not containing die aromatischen Aminosaeuren and Histidine in the protein molecular of MT.The pH value allowing in MT the metal ion of 50% dissociate is respectively: Zn-MT:3.5 ~ 4.5; Cd-MT:2.5 ~ 3.5; Cu-MT<1.0.Because MT lacks die aromatischen Aminosaeuren, therefore at 280nm place without absorption peak, but there is the absorption peak relevant to metal.The MT of metal is gone to have an absorption peak at 190nm place.All MT is contained in all vertebratess, most plants and microbe.Be no matter that natural birth survives be the MT that induction produces, its amino acid composition is substantially identical, main difference is at institute's containing metal and content thereof.Current product mainly extracts from rabbit liver, horse kidney, pork liver and microorganism (as thick neurospora) etc.

About function and the exploitation of metallothionein(MT), successively held four International Workshop symposials altogether in Switzerland, Japan, the U.S. from 1978 to 1999, the 5th International Workshop symposial is held in October, 2005 in Beijing.Within 1987, MT is listed in [863] plan by China, and " eight or five ", " 95 " great brainstorm project, list national level [torch plan] for 1994 in; MT project in 2003 is classified as by the Ministry of Science and Technology that " national science and technology achievement key popularization plan project, nineteen ninety-five, MT was listed in the biological technology products recommended to countries in the world by United Nations.As can be seen here from domestic abroad to the great attention of the research of MT, exploitation, popularization, application.

Biological fermentation about MT albumen is produced, once the genetically engineered recombination method such as useful intestinal bacteria, Bacillus subtilus, suis, yeast, but all puts into production so far because expression amount is too low.

MT4 is mainly being present in human skin tissues.Skin is the organ of human maximum, can be absorbed by contacting skin with the heavy metal of (as in leaded makeup) in pollutent in extraneous dust, various radiation (ultraviolet etc. as sunlight) can cause skin and subcutaneous generation free radical, thus accelerate the collegen filament of skin, spandex fiber aging (by free-radical oxidn) and following the string, oxidized cell aging apoptosis, oxidized DNA easily suddenlys change carcinogenic, therefore, skin is except MT1 and MT2 having whole body to distribute, also emphasis " is deployed troops on garrison duty " MT4, so MT4 has skin histology specificity, it is the important factor of skin heavy metal IPS and delay skin aging, in skin care, delay senility, alleviate in allergic anti-decrepit beauty daily chemical products and have important use.

Summary of the invention

In view of the defect of above expression amount deficiency, main purpose of the present invention be utilize artificial gene complete synthesis method by codon optimized, and expressor head and the tail tandem sequence repeats is become protokaryon (E.coli) expression vector comprising 4 copy MT4 cistrons for 4 times, in order to improve expression amount.

Improve a method for human metal thioalbumen MT4 prokaryotic expression carrier expression amount, comprise and the series connection unit of the coding region containing the human metal thioalbumen molecule MT4 after codon optimized is formed the Tandem repeat comprising 4 copy human metal thioalbumen MT4 cistrons 4 times at prokaryotic expression carrier clone district tandem sequence repeats; By the prokaryotic expression carrier transform both prokaryotic host cell containing Tandem repeat; Cultivate and this cell of amount reproduction, utilize inductor to lead human metal thioalbumen molecule MT4 and express.

The inventive method also can comprise the step utilizing the human metal thioalbumen MT4 of prior art to abduction delivering to carry out separation and purification further.

Wherein, each described series connection unit preferably includes rrna land and the human metal thioalbumen molecule MT4 after codon optimized.

Each described series connection unit also preferably includes the restriction enzyme site being positioned at series connection unit two ends.

MT4 coding region sequence after codon optimized is preferably as shown in SEQIDNO.1.

The tandem sequence repeats region sequence of the MT4 coding region after codon optimized is as shown in SEQIDNO.2.

The coding region gene sequence of the human metal thioalbumen molecule after codon optimized, its nucleotide sequence is as shown in SEQIDNO.1.

For improving a Tandem repeat for human metal thioalbumen MT4 prokaryotic expression amount, described Tandem repeat joins 4 times by unit repeated strings of connecting and forms; Described series connection unit comprises the coding region of rrna land and the human metal thioalbumen MT4 after codon optimized.

Any one in SEQIDNO.5 ~ SEQIDNO.8 of described Tandem repeat sequence preference.

Express a prokaryotic expression carrier of human metal thioalbumen MT4, described prokaryotic expression carrier contains Tandem repeat of the present invention.

The present invention has the following advantages:

1, the optimizing codon of described people MT4 albumen improves the availability of such password in intestinal bacteria.

2, in described MT4 prokaryotic expression carrier, MT4 expresses cistron and adds nearly 3 times, and the average expression amount of MT4 is increased to average 8.9% by 3.4% when singly copying, and expression amount adds 2.6 times;

3, the MT4 albumen that described MT4 prokaryotic expression carrier is expressed is natural unit molecule, and non pregnant women.

Accompanying drawing explanation

Fig. 1 is a kind of MT4 cascaded structure schematic diagram provided by the invention.

Explain: DNA sequence dna representative in schematic diagram is a part for constructed prokaryotic expression carrier, starts, terminate with " T7terminator " with " T7promoter "; " Lacoperator " is lacI binding site; " RBS " is ribosome binding sequence.The transcription termination signal sequence that " T7terminator " is T7 phage.

Embodiment

Below in conjunction with specific embodiment, technical scheme of the present invention is further described, but does not make limitation of the invention.

The technical scheme adopted is as follows:

First, the MT protein molecular of people MT4 totally four kinds of hypotypes is translated into cDNA sequence by the first step of the present invention, then through the codon of DNA2.0 software optimization MTcDNA, it is made to be more suitable for colibacillary codon preference, and composition optimizes is carried out to N-terminal initial 15 amino acid encoding region, avoid the formation of strong secondary structure, the MT4 coding region sequence after codon optimized is preferably as shown in SEQIDNO.1.

Second step is the full length sequence with oligonucleotide substep extension method synthesis MT4,5 ' end adds NcoI restriction enzyme site, 3 ' end adds SpeI---NheI two restriction enzyme sites successively, PCR primer is after NcoI/NheI double digestion purifying, be settled to 50ng/ul, again prokaryotic expression carrier pET28a (+) is cut purifying through NcoI/NheI enzyme, be settled to 100ng/ul.

3rd step, connects and transforms:

MT1b (or MT2a, MT3, MT4) DNA (50ng/ul) 2ul

PET28a (+) carrier DNA (100ng/ul) 2ul

10XT4DNA ligase enzyme damping fluid 1ul

T4DNA ligase enzyme (200U/ul) 1ul

Sterilization deionized water 4ul

1300rpm, centrifugal 5s, mixing, more once centrifugal with method, room temperature leaves standstill connection 2 hours.

Whole 10ul transforms JM109 competence bacteria, and through leaving standstill 30m, heat shock 1m, after adding 1mlLB, after 1h is cultivated in 37 degree of concussions, centrifugal 4m (6000rpm, EP whizzer), abandons supernatant, suspended sediment, is spread evenly across on the soft agar plate containing kantlex, 37 degree of incubator incubated overnight.Next day transfers clone, and PCR identifies positive recombinants, and positive colony, after sequencing analysis is correct, obtains the clone comprising 1 copy MT4 cistron.

Then carrying out first time is connected in series:

Get correct clone 1 strain, do enzyme with two pipes respectively and cut: pipe-1 enzyme SpeI/BamHI is two to be cut, and reclaims large fragment (about 5.5kb) as carrier; Pipe-2 is cut with enzyme XbaI/BamHI is two, reclaims the fragment of 235bps.The recovery product of pipe connecting-1 and pipe-2, after qualification and DNA sequencing are analyzed, obtains the MT4 recombinant expression vector comprising 2 copy MT cistrons.

Second step is connected in series: adopt the first step to be connected in series product (comprising the correct clone of 2 copy MT cistrons): pipe-1 is cut with SpeI/XhoI enzyme, reclaims large fragment (carrier+2 copies MT cistron); Pipe-2 is cut with XbaI/XhoI enzyme, reclaims the DNA fragmentation (comprising 2 copy MT cistrons) of 537bps.Again after connecting qualification, the MT4 prokaryotic expression carrier of 4 copy MT4 cistrons can be obtained, wherein contain the tandem sequence repeats region sequence of the MT4 coding region after codon optimized as shown in SEQIDNO.2.

Shaking flask expression in a small amount:

Get recombinant expression plasmid conversion BL21 (DE3) expression strain of pET28a (+) empty carrier, the mono-copy of MT, 2 copies, 4 copies respectively, picking list bacterium colony inoculates 4ml2XYT substratum (containing 50ug/ml kantlex) respectively, 37 DEG C, 250rpm, concussion is cultivated, until bacterial growth is to logarithmic phase, add 0.4ulIPTG respectively to each pipe, 25 DEG C of inducing culture 6 hours.Collect 1ml bacterium liquid, centrifugation, PBS washes once, suspend with 500ulPBS again, sonicated cells, 12000rpm/15 minute, 4 DEG C centrifugal abandons precipitation, get supernatant loading 20%SDS-PAGE, through Xylene Brilliant Cyanine G colour developing, decolouring, gel imaging instrument calculates the ratio that target stripe accounts for total protein, result (as follows) display increases with copy number, and expression amount increases (according to target ribbon density accounts for total protein density percent than calculating).

Table 1. different MT4 copy number carrier target stripe density under shaking bacterium condition in a small amount accounts for total protein density percent ratio

	Empty carrier	Singly copy plasmid	2 copy plasmids	4 copy plasmids
					Density (%) before correcting	5.2	8.6	9.3	15.1
Density (%) after correcting	0	3.4	4.1	8.9

Claims (9)

1. improve a method for human metal thioalbumen MT4 prokaryotic expression carrier expression amount, it is characterized in that comprising the Tandem repeat series connection unit of the coding region containing the human metal thioalbumen molecule MT4 after codon optimized being comprised 4 copy human metal thioalbumen MT4 cistrons in 4 formation of prokaryotic expression carrier clone district tandem sequence repeats; By the prokaryotic expression carrier transform both prokaryotic host cell containing Tandem repeat; Cultivate and this cell of amount reproduction, utilize inductor to lead human metal thioalbumen molecule MT4 and express.

2. method according to claim 1, is characterized in that each described series connection unit comprises rrna land and the human metal thioalbumen molecule MT4 after codon optimized.

3. method according to claim 1, is characterized in that each described series connection unit also comprises the restriction enzyme site being positioned at series connection unit two ends.

4. method according to claim 1, is characterized in that the MT4 coding region sequence after codon optimized is as shown in SEQIDNO.1.

5. the method according to any one of Claims 1 to 4, is characterized in that the tandem sequence repeats region sequence of the MT4 coding region after codon optimized is as shown in SEQIDNO.2.

6. the coding gene sequence of the human metal thioalbumen molecule after codon optimized, is characterized in that nucleotide sequence is as shown in SEQIDNO.4.

7., for improving a Tandem repeat for human metal thioalbumen MT4 prokaryotic expression amount, it is characterized in that described Tandem repeat joins 4 times by unit repeated strings of connecting and forms; Described series connection unit comprises the coding region of rrna land and the human metal thioalbumen MT4 after codon optimized.

8. Tandem repeat according to claim 7, is characterized in that described tandem sequence repeats region sequence is as shown in SEQIDNO.2.

9. express a prokaryotic expression carrier of human metal thioalbumen MT4, it is characterized in that described prokaryotic expression carrier contains the Tandem repeat described in claim 9 or 10.