CN101812458A - Rachycentron canadus MHC-IIalpha gene sequence - Google Patents

Abstract

The invention discloses a rachycentron canadus MHC-IIalpha gene sequence, the DNA nucleotide thereof and corresponding amino acid sequence are shown as SEQ ID NO. 1. The gene not only lays the foundation for researching action mechanism of fish MHC-IIalpha molecule and relation of MHC-IIalpha gene polymorphism with fish premonition, but also purified protein with biological activity can be obtained by virtue of the gene sequence, and research of novel disease-resistant immune antibody can be implemented.

Description

Rachycentron canadus MHC-IIalpha gene sequence

Technical field

What the present invention relates to is a kind of albumen coded sequence, especially relates to a kind of cabio MHC-II α gene order.

Background technology

MHC (major histocompatibility complex) is a major histocompatibility complex, is meant compound genetic system or the zone with height polymorphism be made up of a series of closely linked gene locus on the karyomit(e).Be that a class cell surface by MHC coding changes membranin, can in conjunction with and present endogenous antigen and exogenous antigen to the T lymphocyte.Different according to chemical structure and function, MHC can be divided into MHCI quasi-molecule and mhc class ii molecule.Wherein, mostly the antigen of II class MHC molecular presentation is the cell exogenous polypeptid, it is assembled into complete tripolymer by α, β and accessory molecule γ chain in endoplasmic reticulum, protease hydrolysis γ chain, antigenic peptide combines with II class MHC molecule, forms steady I I class MHC-complex of polypeptides, is transported to cell surface again, exogenous antigen is assisted T lymphocyte (Th, CD4 with being after the mhc class ii molecule combines to pass in plastosome or lysosome ⁺), thereby trigger immune response.Fish mhc class ii molecule is formed a heterodimer by a chain and β chain with the form of non covalent bond.

The inducing and regulating of MHC wide participation immunne response, the excitating organism specific immune response has very important meaning on immunology.Since MHC have polymorphism, with the dependency and the linkage disequilibrium of disease, therefore, understand the structure and the function of fish mhc gene, seek tumor susceptibility gene or disease resistance gene with the disease-related connection, preventing disease is had very high value.In addition, because the height polymorphism of MHC, thus can be used for population genetics research, and as one of mark of population molecular evolution.Moreover, the polymorphism that MHC has, can be used for the disease-resistant strain of seed selection with the characteristics such as related and linkage disequilibrium of disease.

Summary of the invention

The purpose of this invention is to provide a kind of cabio MHC-II α gene order.

The present invention passes through the degenerate primer with the conserved regions design of the CDS sequence of nearly source species MHC-II α gene, and increases with the PCR method, is cloned into the full expressed sequence of cabio MHC-II α chain gene in conjunction with the RACE technology; By to MHC-II α gene design primers designed effectively, thereby can analyze the tissue distribution situation of this gene, and the expression amount of the MHCII α in the post-stimulatory spleen of LPS is analyzed by quantitative fluorescent PCR.Concrete DNA Nucleotide and amino acid sequence corresponding are as follows:

agtcgactctaagattcacagacgctcagctcagtgacctgctgctgacgaagatgatga 60

><

M M 2

tcaagctgctcctcttcctcccctgtctcatctctgtctctgctgaaagtctccatgtgg 120

Leader?pept?i?de >< α1?domain

I K L L L F L P C P I S V S A E S L H V 22

accttgctatcactggctgttcagactctgatggagaggatatgtactctctggatggag 180

D L A I T G C S D S D G E D M Y S L D G 42

aagagatgtggtttgcagacttcgtcaatcaaaggggagtggagcctcagcccagcttcg 240

E E M W F A D F V N Q R G V E P Q P S F 62

tagatcatattggctacgtgaatggaacttatgaaggagctgtgaccaatcaacagatct 300

V D H I G Y V N G T Y E G A V T N Q Q I 82

gcaggtcaaacctgaagaacagtcgtgtcggtatgaaggacatccccctggaaaatgatc 360

><α2?domain

C R S N L K N S R V G M K D I P L E N D 102

ctccttccacgctcatcatctacagcagagacgaggtggagatcggagaggagaacgtcc 420

P P S T L I I Y S R D E V E I G E E N V 122

tgatctgttacgtgtctgggttctaccctgctcctgtcaaagtccactggaccaagaacg 480

L I C Y V S G F Y P A P V K V H W T K N 142

gagagagcgtgactgagggaacgagtatcaatgttccctatccgaacaaagacggttcct 540

G E S V T E G T S I N V P Y P N K D G S 162

tcaaccagatatccagactgaacttcatcccacagcagggagacatgtacagctgctcag 600

F N Q I S R L N F I P Q Q G D M Y S C S 182

tgacacatccagctctgcaacaacgcatgaccaggatctgggatgtggacgtgcagcagc 660

>< CP/TM/CYT?region

V T H P A L Q Q R M T R I W D V D V Q Q 202

ccagtatcggacctgcggtgttctgtggcctcggtctgacggtcggtttgcttggtgtcg 720

P S I G P A V F C G L G L T V G L L G V 222

cagctggaaccttcttcctcatcaaaggaaacgagtgcagctaattggctggagctgatg 780

A A G T F F L I K G N E C S * 236

atgtcatcagttttttttttttgtatagtgtcctatcaggcttcacctgcttcagagaat 840

cagctgtttatgataatgatgtaaataatcatcttcatcttcatcatctcacttcttaaa 900

aatcactaaaccagatgtcgatgttctgtgatcagctactataaacttcctctttcaaaa 960

taaaagtccagtctcttaatgcaaaaaaaaaaaaaaaa 998

The acquisition of advantage of the present invention: Ben Jiyin not only lays the foundation for the relation of the mechanism of action of researching fish MHC-II α quasi-molecule and MHC-II α gene pleiomorphism and fish body disease resistance, and can obtain the purifying protein of biologically active by gene order, become a reality for studying novel anti pathologic immunity antibody.This gene provides material for fish population genetics and evolutionary genetics research, can be used for population genetics research, and as one of mark of population molecular evolution.Simultaneously can carry out the research of disease-resistant dependency and disease-resistant assistant breeding.

Description of drawings

Fig. 1 is the distribution of the present invention in the cabio healthy tissues;

Fig. 2 is the present invention's MHC-II α gene expression variation diagram in time in the cabio spleen tissue after LPS stimulates.

Embodiment

1. the extraction of total RNA

Get healthy cabio (the about 1000g of body weight) and in culturing bucket, culture after 3 days the shark vibrio of abdominal injection 1ml (JT2).After stimulating 192h, dissect the fish body and take out the about 250mg of spleen, it is standby to put into-80 ℃ of refrigerators.Get the about 100mg of standby tissue and carry out homogenate, extract total RNA according to the test kit explanation with the Trizol (U.S. invitrogen company) that scissors shreds adding 1ml.

2.cDNA first chain is synthetic

Getting the total RNA5 μ of cabio g mixes with reverse transcription primer (oligodT joint primer) 1 μ l (10pmol/L), behind 65 ℃ of heating 5min, place immediately and leave standstill 2min on ice, add 5 * buffer then, the 10mmol/LdNTP mixed solution, Ribonuclease Inhibitor, M-MLV ThermoScript II, reaction system are 20 μ l.Response procedures is 37 ℃, 60min, and it is standby to put into-80 ℃ of preservations at last.

3. design of primers foundation, primer synthetic method

From GenBank, download nearly source species (as people, mouse, rainbow trout etc.) MHCII-α homologous gene CDS sequence, utilize Clustal W software to carry out the multisequencing comparison, determine conserved regions, according to conserved regions sequences Design degenerate primer (J2AF, J2AR), the amplified fragments size is about 324bp.Submitting to English fine horse biotechnology Services Co., Ltd to carry out DNA after the design of primers synthesizes.

4. to the clone of cabio MHC-II α gene cDNA complete sequence

With the degenerate primer (J2AF, J2AR) of the conserved regions design of nearly source species MHC-II α gene C DS sequence, the amplified fragments size is about 324bp.

As template, carry out pcr amplification with degenerate primer with the above-mentioned synthetic first chain cDNA, institute's amplification PCR products detects with 1% agarose gel electrophoresis, and purifying reclaims the purpose product from gel.Then with the PCR product cloning of purifying in the pMD-18T carrier, transformed into escherichia coli TOP 10 competent cells, the picking positive colony extracts plasmid DNA.After degenerated primer PCR detects, will have the segmental plasmid DNA of insertion and carry out two-way order-checking with the M13 universal primer.

According to the cDNA fragment sequence design Auele Specific Primer (RCMC32AOUT, RCMC32AIN, RCMC52AOUT, RCMC52AIN) that has obtained, utilize the terminal rapid amplifying technology of cDNA (Rapid Amplificationof cDNA ends, RACE) to 3 of goal gene ' and 5 ' end carry out pcr amplification.

In 3 ' RACE, utilize half-nest type (semi-nested) PCR method, at first carry out the PCR reaction by outside primer RCMC32AOUT and 3 ' joint primer SMART 3a, products therefrom utilizes inboard Auele Specific Primer RCMC32AIN and 3 ' joint primer SMART 3a to carry out pcr amplification again.

In 5 ' RACE, utilize terminal enzyme (DNA) and dCTP after the cDNA end adds poly (C) tail, with the cDNA behind the tailing as template, utilize outside Auele Specific Primer RCMC52AOUT and OligodG to carry out the pcr amplification first time, gained PCR product utilizes inboard primer RCMC52AIN and OligodG to carry out the pcr amplification second time again.

Resulting PCR product is after carrying out separation detection on 1% the agarose gel electrophoresis, behind the PCR product purification, be cloned in the pMD-18T carrier, transformed into escherichia coli TOP 10 competent cells, the picking positive colony, with the M13 primer plasmid DNA is checked order, order-checking institute calling sequence utilizes DNAStar software to splice with the sequence of degenerate primer amplification gained again.

5. to the mensuration of cabio MHC-II α gene

Resulting PCR product behind the PCR product purification, is cloned in the pMD-18T carrier transformed into escherichia coli TOP10 competent cell, picking positive colony after carrying out separation detection on 1% the agarose gel electrophoresis.Sequencing result carries out homology with BLAST software to be measured, and is defined as cabio MHC-II α dna homolog sequence.

6, the RT-PCR of MHC-II α gene in different tissues analyzes

Get healthy tissues such as cabio (185g) heart, spleen, the gill, head-kidney, muscle, the heart, intestines and brain, the first chain cDNA is a template with the reverse transcription synthetic, application Real-time PCR detection method has carried out studying (β-ActinF to the MHC-II α gene organization expression characteristic of cabio, β-ActinR, J2AF, J2AR).As shown in Figure 1, MHC-II α gene all has expressed in normal cabio tissue, and wherein stronger is expressed in the head-kidney, and the moderate gill, spleen, the brain of being expressed in is in the heart, intestines, muscle a little less than the expression.

7, RT-PCR detects the expression of MHC-II α mRNA

Extraction is through the spleen tissue RNA of the different time points of LPS stimulation, and the first chain cDNA is a template with the reverse transcription synthetic, and expression regulation rule in the post-stimulatory spleen of application Real-time PCR detection LPS (β-ActinF, β-ActinR, J2AF, J2AR).Utilize 2 ^{-Δ Δ CT}Method is calculated relative expression quantity, and as shown in Figure 2, MHC-II α genetic expression presents downward trend after LPS stimulates, and all has significant difference with respect to control group.

The primer that the present invention uses sees the following form:

Sequence table

＜110〉Nanhai Aquatic Inst., Chinese Aquatic Scientific Research Inst

 

＜120〉cabio MHC-II α gene order

 

<160>2

 

<210>1

 

<211>998

 

<212>RNA

 

＜213〉cabio (Rachycentron canadium)

 

<220>

 

<221>3’UTP

 

<222>(765)...(998)

 

<220>

 

<221>5’UTR

 

<222>(1)...(53)

 

<220>

 

<221>CDS

 

<222>(54)...(764)

 

<220>

 

<221>PolyA?site

 

<222>(983)...(998)

 

<400>1

 

agtcgactctaagattcacagacgctcagctcagtgacctgctgctgacgaagatgatga 60

><

M M 2

tcaagctgctcctct?tcctcccctgtctcatctctgtctctgctgaaagtctccatgtgg 120

Leader?peptide >< α1?domain

I K L L L F L P C P I S V S A E S L H V 22

accttgctatcactggctgttcagactctgatggagaggatatgtactctctggatggag 180

D L A I T G C S D S D G E D M Y S L D G 42

aagagatgtggtttgcagacttcgtcaatcaaaggggagtggagcctcagcccagcttcg 240

E E M W F A D F V N Q R G V E P Q P S F 62

tagatcatattggctacgtgaatggaacttatgaaggagctgtgaccaatcaacagatct 300

V D H I G Y V N G T Y E G A V T N Q Q I 82

gcaggtcaaacctgaagaacagtcgtgtcggtatgaaggacatccccctggaaaatgatc 360

><α2?domain

C R S N L K N S R V G M K D I P L E N D 102

ctccttccacgctcatcatctacagcagagacgaggtggagatcggagaggagaacgtcc 420

P P S T L I I Y S R D E V E I G E E N V 122

tgatctgttacgtgtctgggttctaccctgctcctgtcaaagtccactggaccaagaacg 480

L I C Y V S G F Y P A P V K V H W T K N 142

gagagagcgtgactgagggaacgagtatcaatgttccctatccgaacaaagacggttcct 540

G E S V T E G T S I N V P Y P N K D G S 162

tcaaccagatatccagactgaacttcatcccacagcagggagacatgtacagctgctcag 600

F N Q I S R L N F I P Q Q G D M Y S C S 182

tgacacatccagctctgcaacaacgcatgaccaggatctgggatgtggacgtgcagcagc 660

>< CP/TM/CYT?region

V T H P A L Q Q R M T R I W D V D V Q Q 202

ccagtatcggacctgcggtgttctgtggcctcggtctgacggtcggtttgcttggtgtcg 720

P S I G P A V F C G L G L T V G L L G V 222

cagctggaaccttcttcctcatcaaaggaaacgagtgcagctaattggctggagctgatg 780

A A G T F F L I K G N E C S * 236

atgtcatcagttttttttttttgtatagtgtcctatcaggcttcacctgcttcagagaat 840

cagctgtttatgataatgatgtaaataatcatcttcatcttcatcatctcacttcttaaa 900

aatcactaaaccagatgtcgatgttctgtgatcagctactataaacttcctctttcaaaa 960

taaaagtccagtctcttaatgcaaaaaaaaaaaaaaaa 998

 

<210>2

 

<211>236

 

<212>PRT

 

＜213〉cabio (Rachycentron canadium)

 

<400>2

 

MMIKLLLFLPCLISVSAESLHVDLAITGCSDSDGEDMYSLDGEEMWFADFVNQRGVEPQP 60

SFVDHIGYVNGTYEGAVTNQQICRSNLKNSRVGMKDIPLENDPPSTLIIYSRDEVEIGEE 120

NVLICYVSGFYPAPVKVHWTKNGESVTEGTSINVPYPNKDGSFNQISRLNFIPQQGDMYS 180

CSVTHPALQQRMTRIWDVDVQQPSIGPAVFCGLGLTVGLLGVAAGTFFLIKGNECS 236

Claims (1)

1. cabio MHC-II α gene order chain gene sequence is characterized in that: its DNA Nucleotide and amino acid sequence corresponding are as described in the SEQ ID NO.1.

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