CN102154263A - Method for cloning complete sequence of coding region of chick cell retinol binding protein 1 gene - Google Patents

Abstract

The invention discloses a method for cloning complete sequence of a coding region of chick cell retinol binding protein 1 gene. The method comprises the following steps: A1, respectively extracting mRNA (messenger ribonucleic acid) from a plurality of tissues of chick, then carrying out reverse transcription to obtain cDNA (complementary deoxyribonucleic acid), and then making obtained cDNA of each tissue into a cDNA pool; A2, designing primer by taking the mRNA sequence of original chick cell retinol binding protein 1 gene at NCBI (National Center of Biotechnology Information) as a template, amplifying and then carrying out clone sequencing; A3, judging sequences not containing in an amplified fragment according to the mRNA sequence and corresponding positions of the sequences, amplifying part of sequence design primer not amplified by taking the DNA sequence of the gene as a template, transmitting amplified products to biotechnology companies, and then recovering, purifying and sequencing; and A4, splicing sequences obtained in A2 and A3 to obtain the complete cloning region sequence of target gene.

Description

A kind of method of cloning chicken cell retinol conjugated protein 1 gene coding region complete sequence

Technical field

The present invention relates to biological technical field, especially a kind of method of cloning chicken cell retinol conjugated protein 1 gene coding region complete sequence.

Background technology

In functional genomics research, to study, at first should obtain the coding region sequence of this gene, generally carry out common clone or be RACE and obtain and obtain gene coded sequence at present to the function of goal gene.Common clone is low owing to cost, and becomes the prefered method that clone the goal gene coding region in most of laboratories.But for goal gene mRNA reference sequences when imperfect (do not have 5 ' and 3 ' control region, the mRNA reference sequences is the CDS sequence), common clone can not can design primer at two ends and whole C DS region sequence is included (just complete at last mRNA sequence also not necessarily can find the primer of the full coding region sequence that can increase) just, like this can only obtain incomplete CDS sequence.In this case, traditional method be to be RACE and the full CDS sequence that obtains goal gene, but its cost is higher, and the RNA quality of extracting more generally clone have relatively high expectations, so application is less relatively.

Summary of the invention

Technical problem to be solved by this invention provides a kind of method of cloning chicken cell retinol conjugated protein 1 gene coding region complete sequence at the deficiencies in the prior art.

The present invention is by the following technical solutions:

A kind of method of cloning chicken cell retinol conjugated protein 1 gene coding region complete sequence may further comprise the steps: A1, and a plurality of the organizing of chicken extracted the mRNA reverse transcription respectively and become cDNA, then the cDNA of each tissue that obtains is made the cDNA pond; A2, the cellular retinol binding protein 1 gene mRNA sequence that goes up jungle fowl with NCBI is a template design primer, carries out cloning and sequencing after the amplification; A3 judges sequence and the corresponding position that is not included in amplified fragments as yet according to the mRNA sequence, is template with this gene DNA sequence then, increases at the partial sequence of failing to increase design primer, and amplified production send biological worker department to reclaim the purifying order-checking; A4 splices the sequence that A2 and A3 obtain, and just obtains the full coding region sequence of goal gene.

Described method, a plurality of tissues of described chicken comprise the heart, liver, kidney, hypothalamus, uterine tube, ovary, hypophysis.

Described method, primer is SEQ ID NO:2 and SEQ ID NO:3 in the steps A 2.

Described method, primer is SEQ ID NO:4 and SEQ ID NO:5 in the steps A 3.

By crossing the combination of these two kinds of methods, the final clone who realizes the full coding region sequence of goal gene.

The sequence (dash area be CDS sequence) of Fig. 1 for obtaining after the present invention splicing;

Embodiment

Below in conjunction with specific embodiment, the present invention is described in detail.

Concrete implementation step:

(1) the segmental preparation of the extraction of histioid collection, mRNA and cDNA

Get 6 of mountain region, 12w Erlongshan Mountains chickens, after the jugular vein bloodletting, core respectively, liver, kidney, hypothalamus, uterine tube, ovary, seven tissues of hypophysis, place the liquid nitrogen quick-frozen immediately ,-70 ℃ of preservations are standby.

After getting 1-2g respectively and organizing sample to add liquid nitrogen grinding, extract the mRNA of heart, liver, kidney, hypothalamus, ovary, hypophysis, seven tissues of uterine tube with RNAiso Regent, adopt spectrophotometer to detect its concentration and purity qualified (OD260/OD280=1.8-2.0) after, use

Reverse transcription becomes cDNA to PrimeScriptTMRT-PCR Kit with reaction system by the described reaction conditions of its specification sheets, deposits the cDNA mixing (each organizes sub-fraction) of seven tissues standby then.

(2) the pulsating amplification of purpose

1. design of primers: go up cellular retinol binding protein 1 gene mRNA (XM_422635.2) the sequences Design primer (seeing Table 1) of jungle fowl according to NCBI, by primer as can be known, this does not comprise the CDS zone fully to the primer amplification fragment.

Table 1 clone primer sequence

2. amplification system and condition: amplification system (50 μ L): cDNA solution 2.0 μ L (9 tissue cDNA of every chicken are mixed the cDNA pond), upstream primer (10 μ M) 1.5 μ L, downstream primer (10 μ M) 1.5 μ L, Master Mix (2 *) 25 μ L, dH ₂O 20 μ L.Amplification parameter: pre-95 ℃ of 10s of sex change; 95 ℃ of 5s of sex change, 60 ℃ of 30s of renaturation (40 circulations).Product reclaims the purpose segment through 2% agarose gel electrophoresis with Gel Extraction Mini Kit, and-20 ℃ of preservations are standby;

(3) preparation of competent cell

1. with the DH5a bacterial classification streak inoculation kept on the LB solid medium, cultivate 16h-18h for 37 ℃;

2. select the single bacterium colony of fresh DH5a on the LB substratum, be inoculated in the 10mL LB liquid nutrient medium, 37 ℃ of 220r/min acutely shake the bacterium incubated overnight;

3. draw 2mL DH5a bacterium liquid and be inoculated in the Erlenmeyer flask that contains 50mL LB liquid nutrient medium, 37 ℃ of 220r/min acutely shake bacterium and are cultured to OD ₆₀₀=0.4-0.6 (about 2h-4h);

4. will be transferred to 50mL centrifuge tube second month in a season under the bacterial cultures aseptic technique, ice bath 10min;

5. 4 ℃, the centrifugal 10min of 4000r/min abandons supernatant, collects the bacterium colony precipitation;

6. will manage and be inverted 1min, add the 0.1M CaCl of 20mL ice precooling ₂Re-suspended cell dispels with suction pipe, ice bath 30min;

7. once more 4 ℃, the centrifugal 10min of 4000r/min abandons supernatant, collects the bacterium colony precipitation;

8. will manage and be inverted 1min, whenever add the 0.1M CaCl that 50mL bacterium liquid adds the precooling of 2mL ice ₂Resuspended each tube cell is placed in 4 ℃ of refrigerators and is spent the night;

9. add 15% (V/V) ice precooling glycerine by 200 μ L/ parts, the mixing packing ,-70 ℃ are frozen standby.

(4) goal gene and T carrier is connected

1. of short duration centrifugal PMD19-TVector and Control Insert make content be pooled to the pipe end;

Want abundant mixing when 2. using Ligation Mix, dissolving in ice during use at every turn;

3. prepare following dna solution in 0.2mL or 0.5mL centrifuge tube, full dose is 5 μ L:PMD18-T Vector0.5 μ L; Goal gene reclaims product 3 μ L; DdH ₂O mends to 5 μ L;

4. the Ligation Mix that adds 5 μ L;

5. after making it mixing with liquid-transfering gun piping and druming, 16 ℃ of overnight incubation.

(5) conversion of connector

1. get full dose 10 μ L ligation things and add the 1.5mL centrifuge tube that places on the ice face;

2. frozen DH5a competent cell is taken out from-70 ℃ of refrigerators, be placed in the ice bath until melting (about 5min).Vibrate centrifuge tube gently, make it mixing, avoid blowing and beating repeatedly;

3. add 100 μ L competent cells in the 1.5mL centrifuge tube, vibration makes it mixing, ice bath 30min gently;

4. 42 ℃ of water-bath 90s, nonoscillatory;

5. rapidly pipe is moved on in the ice bath, leave standstill 3min;

6. add 500 μ L and be preheated in 37 ℃ the LB liquid nutrient medium, 37 ℃ of gentle vibration 45min (200r/min);

7. draw 200 μ L bacterium liquid and coat on the Amp/LB flat board 37 ℃ of inversions, incubated overnight (16h-24h).

(6) bacterium liquid PCR

Picking colony is inoculated in the 5mL Amp/LB liquid nutrient medium, and 37 ℃ are shaken bacterium and spend the night, and getting 0.5 μ L bacterium liquid is template, carries out PCR.

(7) CDS fails in the district DNA cloning of cloned sequence

1. design of primers: with the goal gene dna sequence dna is template, increases at the sequences Design primer of failing to increase.We know by table 1, and on CRBP1 gene mRNA sequence, clone's the primer still has 42～95 CDS region sequence to fail amplification.And the last jungle fowl dna sequence dna (NC_006096.2) of contrast NCBI is learnt, this section sequence is positioned at chicken CRBP1 exons 1 place just, be positioned between dna sequence dna 42bp～95bp, at a pair of primer of this section sequences Design (seeing Table 2), as shown in Table 2, the primer amplification fragment of design is 189bp (23～211), comprising 42bp～95bp.

2. DNA cloning: reaction conditions: 94 ℃ of pre-sex change 5min → (94 ℃ sex change 30s → 60 ℃ annealing renaturation 30s → 72 ℃ prolong 60s) 35Cycle → 72 ℃ prolong 7min; Amplification system (50 μ L): dna profiling 4.0 μ L, upstream primer (10 μ M) 2.0 μ L, downstream primer (10 μ M) 2.0 μ L, Master Mix (2 *) 25 μ L, dH2O 17.0 μ L.

Table 2DNA amplimer sequence

(8) order-checking and sequence assembly

Send the big genome company of China two-way order-checking step (6) bacterium liquid and step (7) regular-PCR amplified production, use DNAstar software to carry out splicing behind the manual synchronizing sequencing result, obtain the mRNA sequence that chicken CRBP1 gene comprises the 490bp in complete CDS district after the splicing.Through comparison, institute's calling sequence and jungle fowl mRNA sequence identity are 100%.

Should be understood that, for those of ordinary skills, can be improved according to the above description or conversion, and all these improvement and conversion all should belong to the protection domain of claims of the present invention.

Claims (4)

1. a method of cloning chicken cell retinol conjugated protein 1 gene coding region complete sequence is characterized in that, may further comprise the steps: A1, and a plurality of the organizing of chicken extracted the mRNA reverse transcription respectively and become cDNA, then the cDNA of each tissue that obtains is made the cDNA pond; A2, the cellular retinol binding protein 1 gene mRNA sequence that goes up jungle fowl with NCBI is a template design primer, carries out cloning and sequencing after the amplification; A3 judges sequence and the corresponding position that is not included in amplified fragments as yet according to the mRNA sequence, is template with this gene DNA sequence then, increases at the partial sequence of failing to increase design primer, and amplified production send biological worker department to reclaim the purifying order-checking; A4 splices the sequence that A2 and A3 obtain, and just obtains the full coding region sequence of goal gene.

2. method according to claim 1 is characterized in that, a plurality of tissues of described chicken comprise the heart, liver, kidney, hypothalamus, uterine tube, ovary, hypophysis.

3. according to the method for claim 1, it is characterized in that primer is SEQ ID NO:2 and SEQ ID NO:3 in the steps A 2.

4. according to the method for claim 1, it is characterized in that primer is SEQ ID NO:4 and SEQ ID NO:5 in the steps A 3.

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